Compositions and methods for detecting Klebsiella pneumoniae

ABSTRACT

Four highly conserved genes, encoding translation elongation factor Tu, translation elongation factor G, the catalytic subunit of proton-translocating ATPase and the RecA recombinase, are used to generate species-specific, genus-specific, family-specific, group-specific and universal nucleic acid probes and amplification primers to rapidly detect and identify algal, archaeal, bacterial, fungal and parasitical pathogens from clinical specimens for diagnosis. The detection of associated antimicrobial agents resistance and toxin genes are also under the scope of the present invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 11/236,785,filed Sep. 27, 2005, which is a continuation of application Ser. No.10/089,177, filed Mar. 27, 2002, which is the U.S. national phase under35 U.S.C. §371 of prior PCT International Application No.PCT/CA00/01150, filed Sep. 28, 2000, which claims the benefit ofCanadian Application No. 2,307,010 filed May 19, 2000, and CanadianApplication No. 2283458, filed Sep. 28, 1999.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledGENOM.048NPCC2.TXT, created May 24, 2010, which is 1.99 MB in size. Theinformation in the electronic format of the Sequence Listing isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Classical Methods for the Identification of Microorganisms

Microorganisms are classically identified by their ability to utilizedifferent substrates as a source of carbon and nitrogen through the useof biochemical tests such as the API20E™ system (bioMérieux). Forsusceptibility testing, clinical microbiology laboratories use methodsincluding disk diffusion, agar dilution and broth microdilution.Although identifications based on biochemical tests and antibacterialsusceptibility tests are cost-effective, generally two days are requiredto obtain preliminary results due to the necessity of two successiveovernight incubations to identify the bacteria from clinical specimensas well as to determine their susceptibility to antimicrobial agents.There are some commercially available automated systems (i.e. theMicroScan™ system from Dade Behring and the Vitek™ system frombioMérieux) which use sophisticated and expensive apparatus for fastermicrobial identification and susceptibility testing (Stager and Davis,1992, Clin. Microbiol. Rev. 5:302-327). These systems require shorterincubation periods, thereby allowing most bacterial identifications andsusceptibility testing to be performed in less than 6 hours.Nevertheless, these faster systems always require the primary isolationof the bacteria or fungi as a pure culture, a process which takes atleast 18 hours for a pure culture or 2 days for a mixed culture. So, theshortest time from sample reception to identification of the pathogen isaround 24 hours. Moreover, fungi other than yeasts are often difficultor very slow to grow from clinical specimens. Identification must relyon labor-intensive techniques such as direct microscopic examination ofthe specimens and by direct and/or indirect immunological assays.Cultivation of most parasites is impractical in the clinical laboratory.Hence, microscopic examination of the specimen, a few immunologicaltests and clinical symptoms are often the only methods used for anidentification that frequently remains presumptive.

The fastest bacterial identification system, the autoSCAN-Walk-Away™system (Dade Behring) identifies both gram-negative and gram-positivebacterial species from standardized inoculum in as little as 2 hours andgives susceptibility patterns to most antibiotics in 5 to 6 hours.However, this system has a particularly high percentage (i.e. 3.3 to40.5%) of non-conclusive identifications with bacterial species otherthan Enterobacteriaceae (Croizé J., 1995, Lett. Infectiol. 10:109-113;York et al., 1992, J. Clin. Microbiol. 30:2903-2910). ForEnterobacteriaceae, the percentage of non-conclusive identifications was2.7 to 11.4%. The list of microorganisms identified by commercialsystems based on classical identification methods is given in Table 15.

A wide variety of bacteria and fungi are routinely isolated andidentified from clinical specimens in microbiology laboratories. Tables1 and 2 give the incidence for the most commonly isolated bacterial andfungal pathogens from various types of clinical specimens. Thesepathogens are the main organisms associated with nosocomial andcommunity-acquired human infections and are therefore considered themost clinically important.

Clinical Specimens Tested in Clinical Microbiology Laboratories

Most clinical specimens received in clinical microbiology laboratoriesare urine and blood samples. At the microbiology laboratory of theCentre Hospitalier de l'Université Laval (CHUL), urine and blood accountfor approximately 55% and 30% of the specimens received, respectively(Table 3). The remaining 15% of clinical specimens comprise variousbiological fluids including sputum, pus, cerebrospinal fluid, synovialfluid, and others (Table 3). Infections of the urinary tract, therespiratory tract and the bloodstream are usually of bacterial etiologyand require antimicrobial therapy. In fact, all clinical samplesreceived in the clinical microbiology laboratory are tested routinelyfor the identification of bacteria and antibiotic susceptibility.

Conventional Pathogen Identification from Clinical Specimens

Urine Specimens

The search for pathogens in urine specimens is so preponderant in theroutine microbiology laboratory that a myriad of tests have beendeveloped. However, the gold standard remains the classicalsemi-quantitative plate culture method in which 1 μL of urine isstreaked on agar plates and incubated for 18-24 hours. Colonies are thencounted to determine the total number of colony forming units (CFU) perliter of urine. A bacterial urinary tract infection (UTI) is normallyassociated with a bacterial count of 10⁷ CFU/L or more in urine.However, infections with less than 10⁷ CFU/L in urine are possible,particularly in patients with a high incidence of diseases or thosecatheterized (Stark and Maki, 1984, N. Engl. J. Med. 311:560-564).Importantly, approximately 80% of urine specimens tested in clinicalmicrobiology laboratories are considered negative (i.e. bacterial countof less than 10⁷ CFU/L; Table 3). Urine specimens found positive byculture are further characterized using standard biochemical tests toidentify the bacterial pathogen and are also tested for susceptibilityto antibiotics. The biochemical and susceptibility testing normallyrequire 18-24 hours of incubation.

Accurate and rapid urine screening methods for bacterial pathogens wouldallow a faster identification of negative specimens and a more efficienttreatment and care management of patients. Several rapid identificationmethods (Uriscreen™, UTIscreen™, Flash Track™ DNA probes and others)have been compared to slower standard biochemical methods, which arebased on culture of the bacterial pathogens. Although much faster, theserapid tests showed low sensitivities and poor specificities as well as ahigh number of false negative and false positive results (Koening etal., 1992, J. Clin. Microbiol. 30:342-345; Pezzlo et al., 1992, J. Clin.Microbiol. 30:640-684).

Blood Specimens

The Blood Specimens Received In The Microbiology Laboratory Are AlwaysSubmitted For Culture. Blood Culture Systems May Be Manual,Semi-Automated Or Completely Automated. The BACTEC™ System (From BectonDickinson) And The Bactalert™ System (From Organon Teknika Corporation)Are The Two Most Widely Used Automated Blood Culture Systems. TheseSystems Incubate Blood Culture Bottles Under Optimal Conditions ForGrowth Of Most Bacteria. Bacterial Growth Is Monitored Continuously ToDetect Early Positives By Using Highly Sensitive Bacterial GrowthDetectors. Once Growth Is Detected, A Gram Stain Is Performed DirectlyFrom The Blood Culture And Then Used To Inoculate Nutrient Agar Plates.Subsequently, Bacterial Identification And Susceptibility Testing AreCarried Out From Isolated Bacterial Colonies With Automated Systems AsDescribed Previously. Blood Culture Bottles Are Normally Reported AsNegative If No Growth Is Detected After An Incubation Of 6 To 7 Days.Normally, The Vast Majority Of Blood Cultures Are Reported Negative. ForExample, The Percentage Of Negative Blood Cultures At The MicrobiologyLaboratory Of The CHUL For The Period February 1994-January 1995 Was93.1% (Table 3).

Other Clinical Samples

Upon receipt by the clinical microbiology laboratory, all body fluidsother than blood and urine that are from normally sterile sites (i.e.cerebrospinal, synovial, pleural, pericardial and others) are processedfor direct microscopic examination and subsequent culture. Again, mostclinical samples are negative for culture (Table 3). In all thesenormally sterile sites, tests for the universal detection of algae,archaea, bacteria, fungi and parasites would be very useful.

Regarding clinical specimens which are not from sterile sites such assputum or stool specimens, the laboratory diagnosis by culture is moreproblematic because of the contamination by the normal flora. Thebacterial or fungal pathogens potentially associated with the infectionare grown and separated from the colonizing microbes using selectivemethods and then identified as described previously. Of course, theDNA-based universal detection of bacteria would not be useful for thediagnosis of bacterial infections at these non-sterile sites. On theother hand, DNA-based assays for species or genus or family or groupdetection and identification as well as for the detection ofantimicrobial agents resistance genes from these specimens would be veryuseful and would offer several advantages over classical identificationand susceptibility testing methods.

DNA-Based Assays with Any Specimen

There is an obvious need for rapid and accurate diagnostic tests for thedetection and identification of algae, archaea, bacteria, fungi andparasites directly from clinical specimens. DNA-based technologies arerapid and accurate and offer a great potential to improve the diagnosisof infectious diseases (Persing et al., 1993, Diagnostic MolecularMicrobiology: Principles and Applications, American Society forMicrobiology, Washington, D.C.; Bergeron and Ouellette, 1995, Infection23:69-72; Bergeron and Ouellette, 1998, J Clin Microbiol. 36:2169-72).The DNA probes and amplification primers which are objects of thepresent invention are applicable for the detection and identification ofalgae, archaea, bacteria, fungi, and parasites directly from anyclinical specimen such as blood, urine, sputum, cerebrospinal fluid,pus, genital and gastro-intestinal tracts, skin or any other type ofspecimens (Table 3). These assays are also applicable to detection frommicrobial cultures (e.g. blood cultures, bacterial or fungal colonies onnutrient agar, or liquid cell cutures in nutrient broth). The DNA-basedtests proposed in this invention are superior in terms of both rapidityand accuracy to standard biochemical methods currently used for routinediagnosis from any clinical specimens in microbiology laboratories.Since these tests can be performed in one hour or less, they provide theclinician with new diagnostic tools which should contribute to a bettermanagement of patients with infectious diseases. Specimens from sourcesother than humans (e.g. other primates, birds, plants, mammals, farmanimals, livestock, food products, environment such as water or soil,and others) may also be tested with these assays.

A High Percentage of Culture-Negative Specimens

Among all the clinical specimens received for routine diagnosis,approximately 80% of urine specimens and even more (around 95%) forother types of normally sterile clinical specimens are negative for thepresence of bacterial pathogens (Table 3). It would also be desirable,in addition to identify bacteria at the species or genus or family orgroup level in a given specimen, to screen out the high proportion ofnegative clinical specimens with a DNA-based test detecting the presenceof any bacterium (i.e. universal bacterial detection). As disclosed inthe present invention, such a screening test may be based on DNAamplification by PCR of a highly conserved genetic target found in allbacteria. Specimens negative for bacteria would not be amplified by thisassay. On the other hand, those that are positive for any bacteriumwould give a positive amplification signal. Similarly, highly conservedgenes of fungi and parasites could serve not only to identify particularspecies or genus or family or group but also to detect the presence ofany fungi or parasite in the specimen.

Towards the Development of Rapid DNA-Based Diagnostic Tests

A rapid diagnostic test should have a significant impact on themanagement of infections. DNA probe and DNA amplification technologiesoffer several advantages over conventional methods for theidentification of pathogens and antimicrobial agents resistance genesfrom clinical samples (Persing et al., 1993, Diagnostic MolecularMicrobiology: Principles and Applications, American Society forMicrobiology, Washington, D.C.; Ehrlich and Greenberg, 1994, PCR-basedDiagnostics in Infectious Disease, Blackwell Scientific Publications,Boston, Mass.). There is no need for culture of the pathogens, hence theorganisms can be detected directly from clinical samples, therebyreducing the time associated with the isolation and identification ofpathogens. Furthermore, DNA-based assays are more accurate for microbialidentification than currently used phenotypic identification systemswhich are based on biochemical tests and/or microscopic examination.Commercially available DNA-based technologies are currently used inclinical microbiology laboratories, mainly for the detection andidentification of fastidious bacterial pathogens such as Mycobacteriumtuberculosis, Chlamydia trachomatis, Neisseria gonorrhoeae as well asfor the detection of a variety of viruses (Tang Y. and Persing D. H.,Molecular detection and identification of microorganisms, In: P. Murrayet al., 1999, Manual of Clinical Microbiology, ASM press, 7^(th)edition, Washington D.C.). There are also other commercially availableDNA-based assays which are used for culture confirmation assays.

Others have developed DNA-based tests for the detection andidentification of bacterial pathogens which are objects of the presentinvention, for example: Staphylococcus sp. (U.S. Pat. No. 5,437,978),Neisseria sp. (U.S. Pat. No. 5,162,199 and European patent serial no.0,337,896,131) and Listeria monocytogenes (U.S. Pat. Nos. 5,389,513 and5,089,386). However, the diagnostic tests described in these patents arebased either on rRNA genes or on genetic targets different from thosedescribed in the present invention. To our knowledge there are only fourpatents published by others mentioning the use of any of the four highlyconserved gene targets described in the present invention for diagnosticpurposes (PCT international publication number WO92/03455 andWO00/14274, European patent publication number 0 133 671 B1, andEuropean patent publication number 0 133 288 A2). WO92/03455 is focusedon the inhibition of Candida species for therapeutic purposes. Itdescribes antisense oligonucleotide probes hybridizing to Candidamessenger RNA. Two of the numerous mRNA proposed as targets are codingfor translation elongation factor 1 (tef1) and the beta subunit ofATPase. DNA amplification or hybrization are not under the scope oftheir invention and although diagnostic use is briefly mentioned in thebody of the application, no specific claim is made regardingdiagnostics. WO00/14274 describes the use of bacterial recA gene foridentification and speciation of bacteria of the Burkholderia cepaciacomplex. Specific claims are made on a method for obtaining nucleotidesequence information for the recA gene from the target bacteria and afollowing comparison with a standard library of nucleotide sequenceinformation (claim 1), and on the use of PCR for amplification of therecA gene in a sample of interest (claims 4 to 7, and 13). However, theuse of a discriminatory restriction enzyme in a RFLP procedure isessential to fulfill the speciation and WO00/14274 did not mention thatmultiple recA probes could be used simultaneously. Patent EP 0 133 288A2 describes and claims the use of bacterial tuf (and fus) sequence fordiagnostics based on hybridization of a tuf (or fus) probe withbacterial DNA. DNA amplification is not under the scope of EP 0 133 288A2. Nowhere it is mentioned that multiple tuf (or fus) probes could beused simultaneously. No mention is made regarding speciation using tuf(or fus) DNA nucleic acids and/or sequences. The sensitivities of thetuf hybrizations reported are 1×10⁶ bacteria or 1-100 ng of DNA. This ismuch less sensitive than what is achieved by our assays using nucleicacid amplification technologies.

Although there are phenotypic identification methods which have beenused for more than 125 years in clinical microbiology laboratories,these methods do not provide information fast enough to be useful in theinitial management of patients. There is a need to increase the speed ofthe diagnosis of commonly encountered bacterial, fungal and parasiticalinfections. Besides being much faster, DNA-based diagnostic tests aremore accurate than standard biochemical tests presently used fordiagnosis because the microbial genotype (e.g. DNA level) is more stablethan the phenotype (e.g. physiologic level).

Bacteria, fungi and parasites encompass numerous well-known microbialpathogens. Other microorganisms could also be pathogens or associatedwith human diseases. For example, achlorophylious algae of thePrototheca genus can infect humans. Archae, especially methanogens, arepresent in the gut flora of humans (Reeve, J. H., 1999, J. Bacteriol.181:3613-3617). However, methanogens have been associated to pathologicmanifestations in the colon, vagina, and mouth (Belay et al., 1988,Appl. Enviro. Microbiol. 54:600-603; Belay et al., 1990, J. Clin.Microbiol. 28:1666-1668; Weaver et al., 1986, Gut 27:698-704).

In addition to the identification of the infectious agent, it is oftendesirable to identify harmful toxins and/or to monitor the sensitivityof the microorganism to antimicrobial agents. As revealed in thisinvention, genetic identification of the microorganism could beperformed simultaneously with toxin and antimicrobial agents resistancegenes.

Knowledge of the genomic sequences of algal, archaeal, bacterial, fungaland parasitical species continuously increases as testified by thenumber of sequences available from public databases such as GenBank.From the sequences readily available from those public databases, thereis no indication therefrom as to their potential for diagnosticpurposes. For determining good candidates for diagnostic purposes, onecould select sequences for DNA-based assays for (i) the species-specificdetection and identification of commonly encountered bacterial, fungaland parasitical pathogens, (ii) the genus-specific detection andidentification of commonly encountered bacterial, fungal or parasiticalpathogens, (iii) the family-specific detection and identification ofcommonly encountered bacterial, fungal or parasitical pathogens, (iv)the group-specific detection and identification of commonly encounteredbacterial, fungal or parasitical pathogens, (v) the universal detectionof algal, archaeal, bacterial, fungal or parasitical pathogens, and/or(vi) the specific detection and identification of antimicrobial agentsresistance genes, and/or (vii) the specific detection and identificationof bacterial toxin genes. All of the above types of DNA-based assays maybe performed directly from any type of clinical specimens or from amicrobial culture.

In our assigned U.S. Pat. No. 6,001,564 and our WO98/20157 patentpublication, we described DNA sequences suitable for (i) thespecies-specific detection and identification of clinically importantbacterial pathogens, (ii) the universal detection of bacteria, and (iii)the detection of antimicrobial agents resistance genes.

The WO98/20157 patent publication describes proprietary tuf DNAsequences as well as tuf sequences selected from public databases (inboth cases, fragments of at least 100 base pairs), as well asoligonucleotide probes and amplification primers derived from thesesequences. All the nucleic acid sequences described in that patentpublication can enter in the composition of diagnostic kits or productsand methods capable of a) detecting the presence of bacteria and fungib) detecting specifically at the species, genus, family or group levels,the presence of bacteria and fungi and antimicrobial agents resistancegenes associated with these pathogens. However, these methods and kitsneed to be improved, since the ideal kit and method should be capable ofdiagnosing close to 100% of microbial pathogens and associatedantimicrobial agents resistance genes and toxins genes. For example,infections caused by Enterococcus faecium have become a clinical problembecause of its resistance to many antibiotics. Both the detection ofthese bacteria and the evaluation of their resistance profiles aredesirable. Besides that, novel DNA sequences (probes and primers)capable of recognizing the same and other microbial pathogens or thesame and additional antimicrobial agents resistance genes are alsodesirable to aim at detecting more target genes and complement ourearlier patent applications.

The present invention improves the assigned application by disclosingnew proprietary tuf nucleic acids and/or sequences as well as describingnew ways to obtain tuf nucleic acids and/or sequences. In addition wedisclose new proprietary atpD and recA nucleic acids and/or sequences.In addition, new uses of tuf, atpD and recA DNA nucleic acids and/orsequences selected from public databases (Table 11) are disclosed.

Highly Conserved Genes for Identification and Diagnostics

Highly conserved genes are useful for identification of microorganisms.For bacteria, the most studied genes for identification ofmicroorganisms are the universally conserved ribosomal RNA genes (rRNA).Among those, the principal targets used for identification purposes arethe small subunit (SSU) ribosomal 16S rRNA genes (in prokaryotes) and18S rRNA genes (in eukaryotes) (Relman and Persing, Genotyping Methodsfor Microbial Identification, In: D. H. Persing, 1996, PCR Protocols forEmerging Infectious Diseases, ASM Press, Washington D.C.). The rRNAgenes are also the most commonly used targets for universal detection ofbacteria (Chen et al., 1988, FEMS Microbiol. Lett. 57:19-24; McCabe etal., 1999, Mol. Genet. Metabol. 66:205-211) and fungi (Van Burik et al.,1998, J. Clin. Microbiol. 36:1169-1175).

However, it may be difficult to discriminate between closely relatedspecies when using primers derived from the 16S rRNA. In some instances,16S rRNA sequence identity may not be sufficient to guarantee speciesidentity (Fox et al., 1992, Int. J. Syst. Bacteriol. 42:166-170) and ithas been shown that inter-operon sequence variation as well as strain tostrain variation could undermine the application of 16S rRNA foridentification purposes (Clayton et al., 1995, Int. J. Syst. Bacteriol.45:595-599). The heat shock proteins (HSP) are another family of veryconserved proteins. These ubiquitous proteins in bacteria and eukaryotesare expressed in answer to external stress agents. One of the mostdescribed of these HSP is HSP 60. This protein is very conserved at theamino acid level, hence it has been useful for phylogenetic studies.Similar to 16S rRNA, it would be difficult to discriminate betweenspecies using the HSP 60 nucleotide sequences as a diagnostic tool.However, Goh et al. identified a highly conserved region flanking avariable region in HSP 60, which led to the design of universal primersamplifying this variable region (Goh et al., U.S. Pat. No. 5,708,160).The sequence variations in the resulting amplicons were found useful forthe design of species-specific assays.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a specific,ubiquitous and sensitive method using probes and/or amplificationprimers for determining the presence and/or amount of nucleic acids:

-   -   from any algal, archaeal, bacterial, fungal or parasitical        species in any sample suspected of containing said nucleic        acids, and optionally,    -   from specific microbial species or genera selected from the        group consisting of the species or genera listed in Table 4, and        optionally,    -   from an antimicrobial agents resistance gene selected from the        group consisting of the genes listed in Table 5, and optionally,    -   from a toxin gene selected from the group consisting of the        genes listed in Table 6,        -   wherein each of said nucleic acids or a variant or part            thereof comprises a selected target region hybridizable with            said probes or primers;        -   said method comprising the steps of contacting said sample            with said probes or primers and detecting the presence            and/or amount of hybridized probes or amplified products as            an indication of the presence and/or amount of said any            microbial species, specific microbial species or genus or            family or group and antimicrobial agents resistance gene            and/or toxin gene.

In a specific embodiment, a similar method directed to each specificmicrobial species or genus or family or group detection andidentification, antimicrobial agents resistance genes detection, toxingenes detection, and universal bacterial detection, separately, isprovided.

In a more specific embodiment, the method makes use of DNA fragmentsfrom conserved genes (proprietary sequences and sequences obtained frompublic databases), selected for their capacity to sensitively,specifically and ubiquitously detect the targeted algal, archaeal,bacterial, fungal or parasitical nucleic acids.

In a particularly preferred embodiment, oligonucleotides of at least 12nucleotides in length have been derived from the longer DNA fragments,and are used in the present method as probes or amplification primers.To be a good diagnostic candidate, an oligonucleotide of at least 12nucleotides should be capable of hybridizing with nucleic acids fromgiven microorganism(s), and with substantially all strains andrepresentatives of said microorganism(s); said oligonucleotide beingspecies-, or genus-, or family-, or group-specific or universal.

In another particularly preferred embodiment, oligonucleotides primersand probes of at least 12 nucleotides in length are designed for theirspecificity and ubiquity based upon analysis of our databases of tuf,atpD and recA sequences. These databases are generated using bothproprietary and public sequence information. Altogether, these databasesform a sequence repertory useful for the design of primers and probesfor the detection and identification of algal, archaeal, bacterial,fungal and parasitical microorganisms. The repertory can also besubdivided into subrepertories for sequence analysis leading to thedesign of various primers and probes.

The tuf, atpD and recA sequences databases as a product to assist thedesign of oligonucleotides primers and probes for the detection andidentification of algal, archaeal, bacterial, fungal and parasiticalmicroorganisms are also covered.

The proprietary oligonucleotides (probes and primers) are also anotherobject of this invention.

Diagnostic kits comprising probes or amplification primers such as thosefor the detection of a microbial species or genus or family or phylum orgroup selected from the following list consisting of Abiotrophiaadiacens, Acinetobacter baumanii, Actinomycetae, Bacteroides, Cytophagaand Flexibacter phylum, Bacteroides fragilis, Bordetella pertussis,Bordetella sp., Campylobacter jejuni and C. coli, Candida albicans,Candida dubliniensis, Candida glabrata, Candida guilliermondii, Candidakrusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis,Candida zeylanoides, Candida sp., Chlamydia pneumoniae, Chlamydiatrachomatis, Clostridium sp., Corynebacterium sp., Crypococcusneoformans, Cryptococcus sp., Cryptosporidium parvum, Entamoeba sp.,Enterobacteriaceae group, Enterococcuscasseliflavus-flavescens-gallinarum group, Enterococcus faecalis,Enterococcus faecium, Enterococcus gallinarum, Enterococcus sp.,Escherichia coli and Shigella sp. group, Gemella sp., Giardia sp.,Haemophilus influenzae, Klebsiella pneumoniae, Legionella pneumophila,Legionella sp., Leishmania sp., Mycobacteriaceae family, Mycoplasmapneumoniae, Neisseria gonorrhoeae, platelets contaminants group (seeTable 14), Pseudomonas aeruginosa, Pseudomonads group, Staphylococcusaureus, Staphylococcus epidermidis, Staphylococcus haemolyticus,Staphylococcus hominis, Staphylococcus saprophyticus, Staphylococcussp., Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcuspyogenes, Streptococcus sp., Trypanosoma brucei, Trypanosoma cruzi,Trypanosoma sp., Trypanosomatidae family, are also objects of thepresent invention.

Diagnostic kits further comprising probes or amplification primers forthe detection of an antimicrobial agents resistance gene selected fromthe group listed in Table 5 are also objects of this invention.

Diagnostic kits further comprising probes or amplification primers forthe detection of a toxin gene selected from the group listed in Table 6are also objects of this invention.

Diagnostic kits further comprising probes or amplification primers forthe detection of any other algal, archaeal, bacterial, fungal orparasitical species than those specifically listed herein, comprising ornot comprising those for the detection of the specific microbial speciesor genus or family or group listed above, and further comprising or notcomprising probes and primers for the antimicrobial agents resistancegenes listed in Table 5, and further comprising or not comprising probesand primers for the toxin genes listed in Table 6 are also objects ofthis invention.

In a preferred embodiment, such a kit allows for the separate or thesimultaneous detection and identification of the above-listed microbialspecies or genus or family or group; or universal detection of algae,archaea, bacteria, fungi or parasites; or antimicrobial agentsresistance genes; or toxin genes; or for the detection of anymicroorganism (algae, archaea, bacteria, fungi or parasites).

In the above methods and kits, probes and primers are not limited tonucleic acids and may include, but are not restricted to analogs ofnucleotides such as: inosine, 3-nitropyrrole nucleosides (Nichols etal., 1994, Nature 369:492-493), Linked Nucleic Acids (LNA) (Koskin etal., 1998, Tetrahedron 54:3607-3630), and Peptide Nucleic Acids (PNA)(Egholm et al., 1993, Nature 365:566-568).

In the above methods and kits, amplification reactions may include butare not restricted to: a) polymerase chain reaction (PCR), b) ligasechain reaction (LCR), c) nucleic acid sequence-based amplification(NASBA), d) self-sustained sequence replication (3SR), e) stranddisplacement amplification (SDA), f) branched DNA signal amplification(bDNA), g) transcription-mediated amplification (TMA), h) cycling probetechnology (CPT), i) nested PCR, j) multiplex PCR, k) solid phaseamplification (SPA), 1) nuclease dependent signal amplification (NDSA),m) rolling circle amplification technology (RCA), n) Anchored stranddisplacement amplification, o) Solid-phase (immobilized) rolling circleamplification.

In the above methods and kits, detection of the nucleic acids of targetgenes may include real-time or post-amplification technologies. Thesedetection technologies can include, but are not limited to, fluorescenceresonance energy transfer (FRET)-based methods such as adjacenthybridization to FRET probes (including probe-probe and probe-primermethods), TaqMan, Molecular Beacons, scorpions, nanoparticle probes andSunrise (Amplifluor). Other detection methods include target genesnucleic acids detection via immunological methods, solid phasehybridization methods on filters, chips or any other solid support,whether the hybridization is monitored by fluorescence,chemiluminescence, potentiometry, mass spectrometry, plasmon resonance,polarimetry, colorimetry, or scanometry. Sequencing, includingsequencing by dideoxy termination or sequencing by hybridization, e.g.sequencing using a DNA chip, is another possible method to detect andidentify the nucleic acids of target genes.

In a preferred embodiment, a PCR protocol is used for nucleic acidamplification, in diagnostic method as well as in method of constructionof a repertory of nucleic acids and deduced sequences.

In a particularly preferred embodiment, a PCR protocol is provided,comprising, an initial denaturation step of 1-3 minutes at 95° C.,followed by an amplification cycle including a denaturation step of onesecond at 95° C. and an annealing step of 30 seconds at 45-65° C.,without any time allowed specifically for the elongation step. This PCRprotocol has been standardized to be suitable for PCR reactions withmost selected primer pairs, which greatly facilitates the testingbecause each clinical sample can be tested with universal,species-specific, genus-specific, antimicrobial agents resistance geneand toxin gene PCR primers under uniform cycling conditions.Furthermore, various combinations of primer pairs may be used inmultiplex PCR assays.

It is also an object of the present invention that tuf, atpD and recAsequences could serve as drug targets and these sequences and means toobtain them revealed in the present invention can assist the screening,design and modeling of these drugs.

It is also an object of the present invention that tuf, atpD and recAsequences could serve for vaccine purposes and these sequences and meansto obtain them revealed in the present invention can assist thescreening, design and modeling of these vaccines.

We aim at developing a universal DNA-based test or kit to screen outrapidly samples which are free of algal, archaeal, bacterial, fungal orparasitical cells. This test could be used alone or combined with morespecific identification tests to detect and identify the above algaland/or archaeal and/or bacterial and/or fungal and/or parasiticalspecies and/or genera and/or family and/or group and to determinerapidly the bacterial resistance to antibiotics and/or presence ofbacterial toxins. Although the sequences from the selected antimicrobialagents resistance genes are available from public databases and havebeen used to develop DNA-based tests for their detection, our approachis unique because it represents a major improvement over currentdiagnostic methods based on bacterial cultures. Using an amplificationmethod for the simultaneous or independent or sequential microbialdetection-identification and antimicrobial resistance genes detection,there is no need for culturing the clinical sample prior to testing.Moreover, a modified PCR protocol has been developed to detect alltarget DNA sequences in approximately one hour under uniformamplification conditions. This procedure should save lives by optimizingtreatment, should diminish antimicrobial agents resistance because lessantibiotics will be prescribed, should reduce the use of broad spectrumantibiotics which are expensive, decrease overall health care costs bypreventing or shortening hospitalizations, and side effects of drugs,and decrease the time and costs associated with clinical laboratorytesting.

In another embodiment, sequence repertories and ways to obtain them forother gene targets are also an object of this invention, such is thecase for the hexA nucleic acids and/or sequences of Streptococci.

In yet another embodiment, for the detection of mutations associatedwith antibiotic resistance genes, we built repertories to distinguishbetween point mutations reflecting only gene diversity and pointmutations involved in resistance. Such repertories and ways to obtainthem for pbp1a, pbp2b and pbp2x genes of sensitive andpenicillin-resistant Streptoccoccus pneumoniae and also for gyrA andparC gene fragments from various bacterial species are also an object ofthe present invention.

The diagnostic kits, primers and probes mentioned above can be used toidentify algae, archaea, bacteria, fungi, parasites, antimicrobialagents resistance genes and toxin genes on any type of sample, whethersaid diagnostic kits, primers and probes are used for in vitro or insitu applications. The said samples may include but are not limited to:any clinical sample, any environment sample, any microbial culture, anymicrobial colony, any tissue, and any cell line.

It is also an object of the present invention that said diagnostic kits,primers and probes can be used alone or in conjunction with any otherassay suitable to identify microorganisms, including but not limited to:any immunoassay, any enzymatic assay, any biochemical assay, anylysotypic assay, any serological assay, any differential culture medium,any enrichment culture medium, any selective culture medium, anyspecific assay medium, any identification culture medium, anyenumeration culture medium, any cellular stain, any culture on specificcell lines, and any infectivity assay on animals.

In the methods and kits described herein below, the oligonucleotideprobes and amplification primers have been derived from larger sequences(i.e. DNA fragments of at least 100 base pairs). All DNA fragments havebeen obtained either from proprietary fragments or from publicdatabases. DNA fragments selected from public databases are newly usedin a method of detection according to the present invention, since theyhave been selected for their diagnostic potential.

In another embodiment, the amino acid sequences translated from therepertory of tuf, atpD and recA nucleic acids and/or sequences are alsoan object of the present invention.

It is clear to the individual skilled in the art that otheroligonucleotide sequences appropriate for (i) the universal detection ofalgae, archaea, bacteria, fungi or parasites, (ii) the detection andidentification of the above microbial species or genus or family orgroup, and (iii) the detection of antimicrobial agents resistance genes,and (iv) the detection of toxin genes, other than those listed in Tables39-41, 59-60, 70-76, 77-79, and 81-92 may also be derived from theproprietary fragments or selected public database sequences. Forexample, the oligonucleotide primers or probes may be shorter or longerthan the ones chosen; they may also be selected anywhere else in theproprietary DNA fragments or in the sequences selected from publicdatabases; they may be also variants of the same oligonucleotide. If thetarget DNA or a variant thereof hybridizes to a given oligonucleotide,or if the target DNA or a variant thereof can be amplified by a givenoligonucleotide PCR primer pair, the converse is also true; a giventarget DNA may hybridize to a variant oligonucleotide probe or beamplified by a variant oligonucleotide PCR primer. Alternatively, theoligonucleotides may be designed from any DNA fragment sequences for usein amplification methods other than PCR. Consequently, the core of thisinvention is the identification of universal, species-specific,genus-specific, family-specific, group-specific, resistancegene-specific, toxin gene-specific genomic or non-genomic DNA fragmentswhich are used as a source of specific and ubiquitous oligonucleotideprobes and/or amplification primers. Although the selection andevaluation of oligonucleotides suitable for diagnostic purposes requiresmuch effort, it is quite possible for the individual skilled in the artto derive, from the selected DNA fragments, oligonucleotides other thanthe ones listed in Tables 39-41, 59-60, 70-76, 77-79, and 81-92 whichare suitable for diagnostic purposes. When a proprietary fragment or apublic databases sequence is selected for its specificity and ubiquity,it increases the probability that subsets thereof will also be specificand ubiquitous.

Since a high percentage of clinical specimens are negative for bacteria(Table 3), DNA fragments having a high potential for the selection ofuniversal oligonucleotide probes or primers were selected fromproprietary and public database sequences. The amplification primerswere selected from genes highly conserved in algae, archaea, bacteria,fungi and parasites, and are used to detect the presence of any algal,archaeal, bacterial, fungal or parasitical pathogen in clinicalspecimens in order to determine rapidly whether it is positive ornegative for algae, archaea, bacteria, fungi or parasites. The selectedgenes, designated tuf, fus, atpD and recA, encode respectively 2proteins (elongation factors Tu and G) involved in the translationalprocess during protein synthesis, a protein (beta subunit) responsiblefor the catalytic activity of proton pump ATPase and a proteinresponsible for the homologous recombination of genetic material. Thealignments of tuf, atpD and recA sequences used to derive the universalprimers include both proprietary and public database sequences. Theuniversal primer strategy allows the rapid screening of the numerousnegative clinical specimens (around 80% of the specimens received, seeTable 3) submitted for microbiological testing.

Table 4 provides a list of the archaeal, bacterial, fungal andparasitical species for which tuf and/or atpD and/or recA nucleic acidsand/or sequences are revealed in the present invention. Tables 5 and 6provide a list of antimicrobial agents resistance genes and toxin genesselected for diagnostic purposes. Table 7 provides the origin of tuf,atpD and recA nucleic acids and/or sequences listed in the sequencelisting. Tables 8-10 and 12-14 provide lists of species used to test thespecificity, ubiquity and sensitivity of some assays described in theexamples. Table 11 provides a list of microbial species for which tufand/or atpD and/or recA sequences are available in public databases.Table 15 lists the microorganisms identified by commercial systems.Tables 16-18 are part of Example 42, whereas Tables 19-20 are part ofExample 43. Tables 21-22 illustrate Example 44, whereas Tables 23-25illustrate Example 45.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 illustrate the principal subdivisions of the tuf and atpDsequences repertories, respectively. For the design of primers andprobes, depending on the needs, one may want to use the complete dataset illustrated on the top of the pyramid or use only a subsetillustrated by the different branching points. Smaller subdivisions,representing groups, families, genus and species, could even be made toextend to the bottom of the pyramid. Because the tuf and atpD sequencesare highly conserved and evolved with each species, the design ofprimers and probes does not need to include all the sequences within thedatabase or its subdivisions. As illustrated in Tables 42 to 58, 61 to69, 76 and 80, depending on the use, sequences from a limited number ofspecies can be carefully selected to represent: i) only the mainphylogenetic branches from which the intended probes and primers need tobe differentiating, and ii) only the species for which they need to bematching. However, for ubiquity purposes, and especially for primers andprobes identifying large groups of species (genus, family, group oruniversal, or sequencing primers), the more data is included into thesequence analysis, the better the probes and primers will be suitablefor each particular intended use. Similarly, for specificity purposes, alarger data set (or repertory) ensures optimal primers and probes designby reducing the chance of employing nonspecific oligonucleotides.

FIG. 3 illustrates the approach used to design specific amplificationprimers from fusA as well as from the region between the end of fusA andthe beginning of tuf in the streptomycin (str) operon (referred to asthe fusA-tuf intergenic spacer in Table 7). Shown is a schematicorganization of universal amplification primers (SEQ ID NOs. 1221-1229)in the str operon. Amplicon sizes are given in bases pairs. Drawing notto scale, as the fusA-tuf intergenic spacer size varies depending on thebacterial species. Indicated amplicon lengths are for E. coli.

FIGS. 4 to 6 are illustrations to Example 42, whereas FIGS. 7 to 10illustrate Example 43.

FIG. 4. Abridged multiple amino acid sequence alignment of the partialtuf gene products from selected species illustrated using the programAlscript. Residues highly conserved in bacteria are boxed in grey andgaps are represented with dots. Residues in reverse print are unique tothe enterococcal tufB as well as to streptococcal and lactococcal tufgene products. Numbering is based on E. coli EF-Tu and secondarystructure elements of E. coli EF-Tu are represented by cylinders(α-helices) and arrows (β-strands). The sequences shown correspond toSEQ ID NO's: 2630 to 2667.

FIG. 5. Distance matrix tree of bacterial EF-Tu based on amino acidsequence homology. The tree was constructed by the neighbor-joiningmethod. The tree was rooted using archeal and eukaryotic EF-1α genes asthe outgroup. The scale bar represents 5% changes in amino acidsequence, as determined by taking the sum of all of the horizontal linesconnecting two species.

FIG. 6. Southern hybridization of BglII/XbaI digested genomic DNAs ofsome enterococci (except for E. casseliflavus and E. gallinarum whosegenomic DNA was digested with BamHI/PvuII) using the tufA gene fragmentof E. faecium as probes. The sizes of hybridizing fragments are shown inkilobases. Strains tested are listed in Table 16.

FIG. 7. Pantoea and Tatumella species specific signature indel in atpDgenes. The nucleotide positions given are for E. coli atpD sequence(GenBank accession no. V00267). Numbering starts from the first base ofthe initiation codon.

FIG. 8: Trees based on sequence data from tuf (left side) and atpD(right side). The phylogenetic analysis was performed using theNeighbor-Joining method calculated using the Kimura two-parametermethod. The value on each branch indicates the occurence (%) of thebranching order in 750 bootstrapped trees.

FIG. 9: Phylogenetic tree of members of the family Enterobacteriaceaebased on tuf (a), atpD (b), and 16S rDNA (c) genes. Trees were generatedby neighbor-joining method calculated using the Kimura two-parametermethod. The value on each branch is the percentage of bootstrapreplications supporting the branch. 750 bootstrap replications werecalculated.

FIG. 10: Plot of tuf distances versus 16S rDNA distances (a), atpDdistances versus 16S rDNA distances (b), and atpD distances versus tufdistances (c). Symbols: ◯, distances between pairs of strains belongingto the same species; ●, distances between E. coli strains and Shigellastrains; □, distances between pairs belonging to the same genus; ▪,distances between pairs belonging to different genera; Δ, distancesbetween pairs belonging to different families.

FIGS. 11 and 12 are illustrations to Example 44.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present inventors reasoned that comparing the published Haemophilusinfluenzae and Mycoplasma genitalium genomes and searching for conservedgenes could provide targets to develop useful diagnostic primers andprobes. This sequence comparison is highly informative as these twobacteria are distantly related and most genes present in the minimalgenome of M. genitalium are likely to be present in every bacterium.Therefore genes conserved between these two bacteria are likely to beconserved in all other bacteria.

Following the genomic comparison, it was found that severalprotein-coding genes were conserved in evolution. Highly conservedproteins included the translation elongation factors G (EF-G) and Tu(EF-Tu) and the β subunit of F₀F₁ type ATP-synthase, and to a lesserextent, the RecA recombinase. These four proteins coding genes wereselected amongst the 20 most conserved genes on the basis that they allpossess at least two highly conserved regions suitable for the design ofuniversal amplification and sequencing primers. Moreover, within thefragment amplified by these primers, highly conserved and more variableregions are also present hence suggesting it might be possible torapidly obtain sequence information from various microbial species todesign universal as well as species-, genus-, family-, or group-specificprimers and probes of potential use for the detection and identificationand/or quantification of microorganisms.

Translation elongation factors are members of a family of GTP-bindingproteins which intervene in the interactions of tRNA molecules with theribosome machinery during essential steps of protein synthesis. The roleof elongation factor Tu is to facilitate the binding of aminoacylatedtRNA molecules to the A site of the ribosome. The eukaryotic, archaeal(archaebacterial) and algal homolog of EF-Tu is called elongation factor1 alpha (EF-1α). All protein synthesis factors originated from a commonancestor via gene duplications and fusions (Cousineau et al., 1997, J.Mol. Evol. 45:661-670). In particular, elongation factor G (EF-G),although having a functional role in promoting the translocation ofaminoacyl-tRNA molecules from the A site to the P site of the ribosome,shares sequence homologies with EF-Tu and is thought to have arisen fromthe duplication and fusion of an ancestor of the EF-Tu gene.

In addition, EF-Tu is known to be the target for antibiotics belongingto the elfamycin's group as well as to other structural classes (Anborghand Parmeggiani, 1991, EMBO J. 10:779-784; Luiten et al., 1992, Europeanpatent application serial No. EP 0 466 251 A1). EF-G for its part, isthe target of the antibiotic fusidic acid. In addition to its crucialactivities in translation, EF-Tu has chaperone-like functions in proteinfolding, protection against heat denaturation of proteins andinteractions with unfolded proteins (Caldas et al., 1998, J. Biol. Chem.273:11478-11482). Interestingly, a form of the EF-Tu protein has beenidentified as a dominant component of the periplasm of Neisseriagonorrhoeae (Porcella et al., 1996, Microbiology 142:2481-2489), hencesuggesting that at least in some bacterial species, EF-Tu might be anantigen with vaccine potential.

F₀F₁ type ATP-synthase belongs to a superfamily of proton-translocatingATPases divided in three major families: P, V and F (Nelson and Taiz,1989, TIBS 14:113-116). P-ATPases (or E₁-E₂ type) operate via aphosphorylated intermediate and are not evolutionarily related to theother two families. V-ATPases (or V₀V₁ type) are present on the vacuolarand other endomembranes of eukaryotes, on the plasma membrane of archaea(archaebacteria) and algae, and also on the plasma membrane of someeubacteria especially species belonging to the order Spirochaetales aswell as to the Chlamydiaceae and Deinococcaceae families. F-ATPases (orF₀F₁ type) are found on the plasma membrane of most eubacteria, on theinner membrane of mitochondria and on the thylakoid membrane ofchloroplasts. They function mainly in ATP synthesis. They are largemultimeric enzymes sharing numerous structural and functional featureswith the V-ATPases. F and V-type ATPases have diverged from a commonancestor in an event preceding the appearance of eukaryotes. The βsubunit of the F-ATPases is the catalytic subunit and it possesses lowbut significant sequence homologies with the catalytic A subunit ofV-ATPases.

The translation elongation factors EF-Tu, EF-G and EF-1

and the catalytic subunit of F or V-types ATP-synthase, are highlyconserved proteins sometimes used for phylogenetic analysis and theirgenes are also known to be highly conserved (Iwabe et al., 1989, Proc.Natl. Acad. Sci. USA 86:9355-9359, Gogarten et al., 1989, Proc. Natl.Acad. Sci. USA 86:6661-6665, Ludwig et al., 1993, Antonie vanLeeuwenhoek 64:285-305). A recent BLAST (Altschul et al., 1997, J. Mol.Biol. 215:403-410) search performed by the present inventors on theGenBank, European Molecular Biology Laboratory (EMBL), DNA Database ofJapan (DDBJ) and specific genome project databases indicated thatthroughout bacteria, the EF-Tu and the β subunit of F₀F₁ typeATP-synthase genes may be more conserved than other genes that are wellconserved between H. influenzae and M. genitalium.

The RecA recombinase is a multifunctional protein encoded by the recAgene. It plays a central role in homologous recombination, it iscritical for the repair of DNA damage and it is involved in theregulation of the SOS system by promoting the proteolytic digestion ofthe LexA repressor. It is highly conserved in bacteria and could serveas a useful genetic marker to reconstruct bacterial phylogeny (Millerand Kokjohn, 1990, Annu. Rev. Microbiol. 44:365-394). Although RecApossesses some highly conserved sequence segments that we used to designuniversal primers aimed at sequencing the recA fragments, it is clearlynot as well conserved EF-G, EF-Tu and β subunit of F₀F₁ typeATP-synthase. Hence, RecA may not be optimal for universal detection ofbacteria with high sensitivity but it was chosen because preliminarydata indicated that EF-G, EF-Tu and β subunit of F₀F₁ type ATP-synthasemay sometimes be too closely related to find specific primer pairs thatcould discriminate between certain very closely related species andgenera. While RecA, EF-G, EF-Tu and β subunit of F₀F₁ type ATP-synthasegenes, possesses highly conserved regions suitable for the design ofuniversal sequencing primers, the less conserved region between primersshould be divergent enough to allow species-specific and genus-specificprimers in those cases.

Thus, as targets to design primers and probes for the genetic detectionof microorganisms, the present inventors have focused on the genesencoding these four proteins: tuf, the gene for elongation factor Tu(EF-Tu); fus, the gene for the elongation factor G (EF-G); atpD, thegene for β subunit of F₀F₁ type ATP-synthase; and recA, the geneencoding the RecA recombinase. In several bacterial genomes tuf is oftenfound in two highly similar duplicated copies named tufA and tufB (Filerand Furano, 1981, J. Bacteriol. 148:1006-1011, Sela et al., 1989, J.Bacteriol. 171:581-584). In some particular cases, more divergent copiesof the tuf genes can exist in some bacterial species such as someactinomycetes (Luiten et al. European patent application publication No.EP 0 446 251 A1; Vijgenboom et al., 1994, Microbiology 140:983-998) and,as revealed as part of this invention, in several enterococcal species.In several bacterial species, tuf is organized in an operon with itshomolog gene for the elongation factor G (EF-G) encoded by the fusA gene(FIG. 3). This operon is often named the str operon. The tuf, fus, atpDand recA genes were chosen as they are well conserved in evolution andhave highly conserved stretches as well as more variable segments.Moreover, these four genes have eukaryotic orthologs which are describedin the present invention as targets to identify fungi and parasites. Theeukaryotic homolog of elongation factor Tu is called elongation factor1-alpha (EF-1α) (gene name: tef, teff, eft, ef-1 or EF-1). In fungi, thegene for EF-1α occurs sometimes in two or more highly similar duplicatedcopies (often named tef1, tef2, tef3 . . . ). In addition, eukaryoteshave a copy of elongation factor Tu which is originating from theirorganelle genome ancestry (gene name: tuf1, tufM or tufA). For thepurpose of the current invention, the genes for these four functionallyand evolutionarily linked elon-gation factors (bacterial EF-Tu and EF-G,eukaryotic EF-1α, and organellar EF-Tu) will hereafter be designated as<<tuf nucleic acids and/or sequences>>. The eukaryotic (mitochondrial)F₀F₁ type ATP-synthase beta subunit gene is named atp2 in yeast. For thepurpose of the current invention, the genes of catalytic sub-unit ofeither F or V-type ATP-synthase will hereafter be designated as <<atpDnucleic acids and/or sequences>>. The eukaryotic homologs of RecA aredistributed in two families, typified by the Rad51 and Dmc1 proteins.Archaeal homologs of RecA are called RadA. For the purpose of thecurrent invention, the genes corres-ponding to the latter proteins willhereafter be designated as <<recA nucleic acids and/or sequences>>.

In the description of this invention, the terms <<nucleic acids>> and<<sequences>> might be used interchangeably. However, <<nucleic acids>>are chemical entities while <<sequences>> are the pieces of informationderived from (inherent to) these <<nucleic acids>>. Both nucleic acidsand sequences are equiva-lently valuable sources of information for thematter pertaining to this invention.

Analysis of multiple sequence alignments of tuf and atpD sequencespermitted the design of oligonucleotide primers (and probes) capable ofamplifying (or hybridizing to) segments of tuf (and/or fus) and atpDgenes from a wide variety of bacterial species (see Examples 1 to 4, 24and 26, and Table 7). Sequencing and amplification primer pairs for tufnucleic acids and/or sequences are listed in Annex I and hybridizationprobes are listed in Tables 41 and 85. Sequencing and amplificationprimer pairs for atpD nucleic acids and/or sequences are listed in Table40. Analysis of the main subdivisions of tuf and atpD sequences (seeFIGS. 1 and 2) permitted to design sequencing primers amplifyingspecifically each of these subdivisions. It should be noted that thesesequencing primers could also be used as universal primers. However,since some of these sequencing primers include several variable sequence(degenerated) positions, their sensitivity could be lower than that ofuniversal primers developed for diagnostic purposes. Furthersubdivisions could be done on the basis of the various phyla where thesegenes are encountered.

Similarly, analysis of multiple sequence alignments of recA sequencespresent in the public databases permitted the design of oligonucleotideprimers capable of amplifying segments of recA genes from a wide varietyof bacterial species. Sequencing and amplification primer pairs for recAsequences are listed in Table 59. The main subdivisions of recA nucleicacids and/or sequences comprise recA, radA, rad51 and dmc1. Furthersubdivisions could be done on the basis of the various phyla where thesegenes are encountered.

The present inventor's strategy is to get as much sequence datainformation from the four conserved genes (tuf, fus, atpD and recA).This ensemble of sequence data forming a repertory (with subrepertoriescorresponding to each target gene and their main sequence subdivisions)and then using the sequence information of the sequence repertory (orsubrepertories) to design primer pairs that could permit eitheruniversal detection of algae or archaea or bacteria or fungi orparasites, detection of a family or group of microorganism (e.g.Enterobacteriaceae), detection of a genus (e.g. Streptococcus) orfinally a specific species (e.g. Staphylococcus aureus). It should benoted that for the purpose of the present invention a group ofmicroorganisms is defined depending on the needs of the particulardiagnostic test. It does not need to respect a particular taxonomicalgrouping or phylum. See Example 12 where primers were designed toamplify a group a bacteria consisting of the 17 major bacterial speciesencountered as contaminants of platelet concentrates. Also remark thatin that Example, the primers are not only able to sensitively andrapidly detect at least the 17 important bacterial species, but couldalso detect other species as well, as shown in Table 14. In thesecircumstances the primers shown in Example 12 are considered universalfor platelet-contaminating bacteria. To develop an assay specific forthe latter, one or more primers or probes specific to each species couldbe designed. Another example of primers and/or probes for groupdetection is given by the Pseudomonad group primers. These primers weredesigned based upon alignment of tuf sequences from real Pseudomonasspecies as well as from former Pseudomonas species such asStenotrophomonas maltophilia. The resulting primers are able to amplifyall Pseudomonas species tested as well as several species belonging todifferent genera, hence as being specific for a group includingPseudomonas and other species, we defined that group as Pseudomonads, asseveral members were former Pseudomonas.

For certain applications, it may be possible to develop a universal,group, family or genus-specific reaction and to proceed to speciesidentification using sequence information within the amplicon to designspecies-specific internal probes or primers, or alternatively, toproceed directly by sequencing the amplicon. The various strategies willbe discussed further below.

The ensembles formed by public and proprietary tuf, atpD and recAnucleic acids and/or sequences are used in a novel fashion so theyconstitute three databases containing useful information for theidentification of microorganisms.

Sequence repertories of other gene targets were also built to solve somespecific identification problems especially for microbial speciesgenetically very similar to each other such as E. coli and Shigella (seeExample 23). Based on tuf, atpD and recA sequences, Streptococcuspneumoniae is very difficult to differentiate from the closely relatedspecies S. oralis and S. mitis. Therefore, we elected to built asequence repertory from hexA sequences (Example 19), a gene much morevariable than our highly conserved tuf, atpD and recA nucleic acidsand/or sequences.

For the detection of mutations associated with antibiotic resistancegenes, we also built repertories to distinguish between point mutationsreflecting only gene diversity and point mutations involved inresistance. This was done for pbp1a, pbp2b and pbp2x genes ofpenicillin-resistant and sensitive Streptoccoccus pneumoniae (Example18) and also for gyrA and parC gene fragments of various bacterialspecies for which quinolone resistance is important to monitor.

Oligonucleotide Primers and Probes Design and Synthesis

The tuf, fus, atpD and recA DNA fragments sequenced by us and/orselected from public databases (GenBank and EMBL) were used to designoligonucleotides primers and probes for diagnostic purposes. Multiplesequence alignments were made using subsets of the tuf or atpD or recAsequences repertory. Subsets were chosen to encompass as much aspossible of the targetted microorganism(s) DNA sequence data and alsoinclude sequence data from phylogenetically related microorganisms fromwhich the targetted microorganism(s) should be distinguished. Regionssuitable for primers and probes should be conserved for the targettedmicroorganism(s) and divergent for the microorganisms from which thetargetted microorganism(s) should be distinguished. The large amount oftuf or atpD or recA sequences data in our repertory permits to reducetrial and errors in obtaining specific and ubiquitous primers andprobes. We also relied on the corresponding peptide sequences of tuf,fus, atpD and recA nucleic acids and/or sequences to facilitate theidentification of regions suitable for primers and probes design. Aspart of the design rules, all oligonucleotides (probes for hybridizationand primers for DNA amplification by PCR) were evaluated for theirsuitability for hybridization or PCR amplification by computer analysisusing standard programs (i.e. the Genetics Computer Group (GCG) programsand the primer analysis software Oligo™ 5.0). The potential suitabilityof the PCR primer pairs was also evaluated prior to the synthesis byverifying the absence of unwanted features such as long stretches of onenucleotide and a high proportion of G or C residues at the 3′ end(Persing et al., 1993, Diagnostic Molecular Microbiology: Principles andApplications, American Society for Microbiology, Washington, D.C.).Oligonucleotide probes and amplification primers were synthesized usingan automated DNA synthesizer (Perkin-Elmer Corp., Applied Bio systemsDivision).

The oligonucleotide sequence of primers or probes may be derived fromeither strand of the duplex DNA. The primers or probes may consist ofthe bases A, G, C, or T or analogs and they may be degenerated at one ormore chosen nucleotide position(s). The primers or probes may be of anysuitable length and may be selected anywhere within the DNA sequencesfrom proprietary fragments or from selected database sequences which aresuitable for (i) the universal detection of algae or archaea or bacteriaor fungi or parasites, (ii) the species-specific detection andidentification of any microorganism, including but not limited to:Abiotrophia adiacens, Bacteroides fragilis, Bordetella pertussis,Candida albicans, Candida dubliniensis, Candida glabrata, Candidaguilliermondii, Candida krusei, Candida lusitaniae, Candidaparapsilosis, Candida tropicalis, Candida zeylanoides, Campylobacterjejuni and C. coli, Chlamydia pneumoniae, Chlamydia trachomatis,Cryptococcus neoformans, Cryptosporidium parvum, Enterococcus faecalis,Enterococcus faecium, Enterococcus gallinarum, Escherichia coli,Haemophilus influenzae, Legionella pneumophila, Mycoplasma pneumoniae,Neisseria gonorrhoeae, Pseudomonas aeruginosa, Staphylococcus aureus,Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcushominis, Staphylococcus saprophyticus, Streptococcus agalactiae,Streptococcus pneumoniae, Trypanosoma brucei, Trypanosoma cruzi, (iii)the genus-specific detection of Bordetella species, Candida species,Clostridium species, Corynebacterium species, Cryptococcus species,Entamoeba species, Enterococcus species, Gemella species, Giardiaspecies, Legionella species, Leishmania species, Staphylococcus species,Streptococcus species, Trypanosoma species, (iv) the family-specificdetection of Enterobacteriaceae family members, Mycobacteriaceae familymembers, Trypanosomatidae family members, (v) the detection ofEnterococcus casseliflavus-flavescens-gallinarum group, Enterococcus,Gemella and Abiotrophia adiacens group, Pseudomonads extended group,Platelet-contaminating bacteria group, (vi) the detection of clinicallyimportant antimicrobial agents resistance genes listed in Table 5, (vii)the detection of clinically important toxin genes listed in Table 6.

Variants for a given target microbial gene are naturally occurring andare attributable to sequence variation within that gene during evolution(Watson et al., 1987, Molecular Biology of the Gene, 4^(th) ed., TheBenjamin/Cummings Publishing Company, Menlo Park, Calif.; Lewin, 1989,Genes IV, John Wiley & Sons, New York, N.Y.). For example, differentstrains of the same microbial species may have a single or morenucleotide variation(s) at the oligonucleotide hybridization site. Theperson skilled in the art is well aware of the existence of variantalgal, archaeal, bacterial, fungal or parasitical DNA nucleic acidsand/or sequences for a specific gene and that the frequency of sequencevariations depends on the selective pressure during evolution on a givengene product. The detection of a variant sequence for a region betweentwo PCR primers may be demonstrated by sequencing the amplificationproduct. In order to show the presence of sequence variants at theprimer hybridization site, one has to amplify a larger DNA target withPCR primers outside that hybridization site. Sequencing of this largerfragment will allow the detection of sequence variation at this site. Asimilar strategy may be applied to show variants at the hybridizationsite of a probe. Insofar as the divergence of the target nucleic acidsand/or sequences or a part thereof does not affect the specificity andubiquity of the amplification primers or probes, variant microbial DNAis under the scope of this invention. Variants of the selected primersor probes may also be used to amplify or hybridize to a variant DNA.

Sequencing of tuf Nucleic Acids and/or Sequences from a Variety ofArchaeal, Bacterial, Fungal and Parasitical Species

The nucleotide sequence of a portion of tuf nucleic acids and/orsequences was determined for a variety of archaeal, bacterial, fungaland parasitical species. The amplification primers (SEQ ID NOs. 664 and697), which amplify a tuf gene portion of approximately 890 bp, wereused along with newly designed sequencing primer pairs (See Annex I forthe sequencing primers for tuf nucleic acids and/or sequences). Mostprimer pairs can amplify different copies of tuf genes (tufA and tufB).This is not surprising since it is known that for several bacterialspecies these two genes are nearly identical. For example, the entiretufA and tufB genes from E. coli differ at only 13 nucleotide positions(Neidhardt et al., 1996, Escherichia coli and Salmonella: Cellular andMolecular Biology, 2^(nd) ed., American Society for Microbiology Press,Washington, D.C.). Similarly, some fungi are known to have two nearlyidentical copies of tuf nucleic acids and/or sequences (EF-1□). Theseamplification primers are degenerated at several nucleotide positionsand contain inosines in order to allow the amplification of a wide rangeof tuf nucleic acids and/or sequences. The strategy used to select theseamplification primers is similar to that illustrated in Table 39 for theselection of universal primers. The tuf sequencing primers evensometimes amplified highly divergent copies of tuf genes (tufC) asillustrated in the case of some enterococcal species (SEQ ID NOs.: 73,75, 76, 614 to 618, 621 and 987 to 989). To prove this, we havedetermined the enterococcal tuf nucleic acids and/or sequences from PCRamplicons cloned into a plasmid vector. Using the sequence data from thecloned amplicons, we designed new sequencing primers specific to thedivergent (tufC) copy of enterococci (SEQ ID NOs.: 658-659 and 661) andthen sequenced directly the tufC amplicons. The amplification primers(SEQ ID NOs.: 543, 556, 557, 643-645, 660, 664, 694, 696 and 697) couldbe used to amplify the tuf nucleic acids and/or sequences from anybacterial species. The amplification primers (SEQ ID NOs.: 558, 559,560, 653, 654, 655, 813, 815, 1974-1984, 1999-2003) could be used toamplify the tuf (EF-1□ genes from any fungal and/or parasitical species.The amplification primers SEQ ID NOs. 1221-1228 could be used to amplifybacterial tuf nucleic acids and/or sequences of the EF-G subdivision(fusA) (FIG. 3). The amplification primers SEQ ID NOs. 1224, and1227-1229 could be used to amplify bacterial tuf nucleic acids and/orsequences comprising the end of EF-G (fusA) and the beginning of EF-Tu(tuf), including the intergenic region, as shown in FIG. 3.

Most tuf fragments to be sequenced were amplified using the followingamplification protocol: One μl of cell suspension (or of purifiedgenomic DNA 0.1-100 ng/μl) was transferred directly to 19 μl of a PCRreaction mixture. Each PCR reaction contained 50 mM KCl, 10 mM Tris-HCl(pH 9.0), 0.1% Triton X-100, 2.5 mM MgCl₂, 1 μM of each of the 2primers, 200 μM of each of the four dNTPs, 0.5 unit of Taq DNApolymerase (Promega Corp., Madison, Wis.). PCR reactions were subjectedto cycling using a PTC-200 thermal cycler (MJ Research Inc., Watertown,Mass.) as follows: 3 min at 94-96° C. followed by 30-45 cycles of 1 minat 95° C. for the denaturation step, 1 min at 50-55° C. for theannealing step and 1 min at 72° C. for the extension step. Subsequently,twenty microliters of the PCR-amplified mixture were resolved byelectrophoresis in a 1.5% agarose gel. The amplicons were thenvisualized by staining with methylene blue (Flores et al., 1992,Biotechniques, 13:203-205). The size of the amplification products wasestimated by comparison with a 100-bp molecular weight ladder. The bandcorresponding to the specific amplification product was excised from theagarose gel and purified using the QIAquick™ gel extraction kit (QIAGENInc., Chatsworth, Calif.). The gel-purified DNA fragment was then useddirectly in the sequencing protocol. Both strands of the tuf genesamplification product were sequenced by the dideoxynucleotide chaintermination sequencing method by using an Applied Biosystems automatedDNA sequencer (model 377) with their Big Dye™ Terminator CycleSequencing Ready Reaction Kit (Applied Biosystems, Foster City, Calif.).The sequencing reactions were performed by using the same amplificationprimers and 10 ng/100 bp of the gel-purified amplicon per reaction. Forthe sequencing of long amplicons such as those of eukaryotic tuf (EF-1

nucleic acids and/or sequences, we designed internal sequencing primers(SEQ ID NOs.: 654, 655 and 813) to be able to obtain sequence data onboth strands for most of the fragment length. In order to ensure thatthe determined sequence did not contain errors attributable to thesequencing of PCR artefacts, we have sequenced two preparations of thegel-purified tuf amplification product originating from two independentPCR amplifications. For most target microbial species, the sequencesdetermined for both amplicon preparations were identical. In case ofdiscrepancies, amplicons from a third independent PCR amplification weresequenced. Furthermore, the sequences of both strands were 100%complementary thereby confirming the high accuracy of the determinedsequence. The tuf nucleic acids and/or sequences determined using theabove strategy are described in the Sequence Listing. Table 7 gives theoriginating microbial species and the source for each tuf sequence inthe Sequence Listing.

The alignment of the tuf sequences determined by us or selected fromdatabases revealed clearly that the length of the sequenced portion ofthe tuf genes is variable. There may be insertions or deletions ofseveral amino acids. In addition, in several fungi introns wereobserved. Intron nucleic acids and/or sequences are part of tuf nucleicacids and/or sequences and could be useful in the design ofspecies-specific primers and probes. This explains why the size of thesequenced tuf amplification products was variable from one fungalspecies to another. Consequently, the nucleotide positions indicated ontop of each of Tables 42 to 58, 61 to 69, 76 and 80 do not correspondfor sequences having insertions or deletions.

It should also be noted that the various tuf nucleic acids and/orsequences determined by us occasionally contain base ambiguities. Thesedegenerated nucleotides correspond to sequence variations between tufAand tufB genes (or copies of the EF-G subdivision of tuf nucleic acidsand/or sequences, or copies of EF-10 subdivision of tuf nucleic acidsand/or sequences for fungi and parasites) because the amplificationprimers amplify both tuf genes. These nucleotide variations were notattributable to nucleotide misincorporations by the Taq DNA polymerasebecause the sequence of both strands was identical and also because thesequences determined with both preparations of the gel-purified tufamplicons obtained from two independent PCR amplifications wereidentical.

The Selection of Amplification Primers from tuf Nucleic Acids and/orSequences

The tuf sequences determined by us or selected from public databaseswere used to select PCR primers for universal detection of bacteria, aswell as for genus-specific, species-specific family-specific orgroup-specific detection and identification. The strategy used to selectthese PCR primers was based on the analysis of multiple sequencealignments of various tuf sequences. For more details about theselection of PCR primers from tuf sequences please refer to Examples 5,7-14, 17, 22, 24, 28, 30-31, 33, 36, and 38-40, and to Tables 44-47,49-57 and 63.

Sequencing of atpD and recA Nucleic Acids and/or Sequences from aVariety of Archaeal, Bacterial, Fungal and Parasitical Species

The method used to obtain atpD and recA nucleic acids and/or sequencesis similar to that described above for tuf nucleic acids and/orsequences.

The Selection of Amplification Primers from atpD or recA Nucleic Acidsand/or Sequences

The comparison of the nucleotide sequence for the atpD or recA genesfrom various archaeal, bacterial, fungal and parasitical species allowedthe selection of PCR primers (refer to Examples 6, 13, 29, 34 and 37,and to Tables 42, V43, 48, and 58).

DNA Amplification

For DNA amplification by the widely used PCR (polymerase chain reaction)method, primer pairs were derived from proprietary DNA fragments or fromdatabase sequences. Prior to synthesis, the potential primer pairs wereanalyzed by using the Oligo™ 5.0 software to verify that they were goodcandidates for PCR amplification.

During DNA amplification by PCR, two oligonucleotide primers bindingrespectively to each strand of the heat-denatured target DNA from themicrobial genome are used to amplify exponentially in vitro the targetDNA by successive thermal cycles allowing denaturation of the DNA,annealing of the primers and synthesis of new targets at each cycle(Persing et al, 1993, Diagnostic Molecular Microbiology: Principles andApplications, American Society for Microbiology, Washington, D.C.).

Briefly, the PCR protocols were as follows: Treated clinical specimensor standardized bacterial or fungal or parasitical suspensions (seebelow) or purified genomic DNA from bacteria, fungi or parasites wereamplified in a 20 μl PCR reaction mixture. Each PCR reaction contained50 mM KCl, 10 mM Tris-HCl (pH 9.0), 2.5 mM MgCl₂, 0.4 μM of each primer,200 μM of each of the four dNTPs and 0.5 unit of Taq DNA polymerase(Promega) combined with the TaqStart™ antibody (Clontech LaboratoriesInc., Palo Alto, Calif.). The TaqStart™ antibody, which is aneutralizing monoclonal antibody to Taq DNA polymerase, was added to allPCR reactions to enhance the specificity and the sensitivity of theamplifications (Kellogg et al., 1994, Biotechniques 16:1134-1137). Thetreatment of the clinical specimens varies with the type of specimentested, since the composition and the sensitivity level required aredifferent for each specimen type. It consists in a rapid protocol tolyse the microbial cells and eliminate or neutralize PCR inhibitors. Foramplification from bacterial or fungal or parasitical cultures or frompurified genomic DNA, the samples were added directly to the PCRamplification mixture without any pre-treatment step. An internalcontrol was derived from sequences not found in the targetmicroorganisms or in the human genome. The internal control wasintegrated into all amplification reactions to verify the efficiency ofthe PCR assays and to ensure that significant PCR inhibition was absent.Alternatively, an internal control derived from rRNA was also useful tomonitor the efficiency of microbial lysis protocols.

PCR reactions were then subjected to thermal cycling (3 min at 94-96° C.followed by 30 cycles of 1 second at 95° C. for the denaturation stepand 30 seconds at 50-65° C. for the annealing-extension step) using aPTC-200 thermal cycler (MJ Research Inc.). The number of cyclesperformed for the PCR assays varies according to the sensitivity levelrequired. For example, the sensitivity level required for microbialdetection directly from clinical specimens is higher for blood specimensthan for urine specimens because the concentration of microorganismsassociated with a septicemia can be much lower than that associated witha urinary tract infection. Consequently, more sensitive PCR assayshaving more thermal cycles are probably required for direct detectionfrom blood specimens. Similarly, PCR assays performed directly frombacterial or fungal or parasitical cultures may be less sensitive thanPCR assays performed directly from clinical specimens because the numberof target organisms is normally much lower in clinical specimens than inmicrobial cultures.

The person skilled in the art of DNA amplification knows the existenceof other rapid amplification procedures such as ligase chain reaction(LCR), transcription-mediated amplification (TMA), self-sustainedsequence replication (3SR), nucleic acid sequence-based amplification(NASBA), strand displacement amplification (SDA), branched DNA (bDNA),cycling probe technology (CPT), solid phase amplification (SPA), rollingcircle amplification technology (RCA), solid phase RCA, anchored SDA andnuclease dependent signal amplification (NDSA) (Lee et al., 1997,Nucleic Acid Amplification Technologies: Application to DiseaseDiagnosis, Eaton Publishing, Boston, Mass.; Persing et al., 1993,Diagnostic Molecular Microbiology: Principles and Applications, AmericanSociety for Microbiology, Washington, D.C.; Westin et al., 2000, Nat.Biotechnol. 18:199-204). The scope of this invention is not limited tothe use of amplification by PCR, but rather includes the use of anyrapid nucleic acid amplification method or any other procedure which maybe used to increase the sensitivity and/or the rapidity of nucleicacid-based diagnostic tests. The scope of the present invention alsocovers the use of any nucleic acids amplification and detectiontechnology including real-time or post-amplification detectiontechnologies, any amplification technology combined with detection, anyhybridization nucleic acid chips or arrays technologies, anyamplification chips or combination of amplification and hybridizationchips technologies. Detection and identification by any sequencingmethod is also under the scope of the present invention.

Any oligonucleotide suitable for the amplification of nucleic acids byapproaches other than PCR or for DNA hybridization which are derivedfrom the species-specific, genus-specific and universal DNA fragments aswell as from selected antimicrobial agents resistance or toxin genesequences included in this document are also under the scope of thisinvention.

Detection of Amplification Products

Classically, detection of amplification is performed by standardethidium bromide-stained agarose gel electrophoresis. It is clear thatother methods for the detection of specific amplification products,which may be faster and more practical for routine diagnosis, may beused. Such methods may be based on the detection of fluorescence afteror during amplification. One simple method for monitoring amplified DNAis to measure its rate of formation by measuring the increase influorescence of intercalating agents such as ethidium bromide or SYBR®Green I (Molecular Probes). If more specific detection is required,fluorescence-based technologies can monitor the appearance of a specificproduct during the reaction. The use of dual-labeled fluorogenic probessuch as in the TaqMan™ system (Applied Biosystems) which utilizes the5′-3′ exonuclease activity of the Taq polymerase is a good example(Livak K. J. et al. 1995, PCR Methods Appl. 4:357-362). TaqMan™ can beperformed during amplification and this “real-time” detection can bedone in a single closed tube hence eliminating post-PCR sample handlingand consequently preventing the risk of amplicon carryover. Severalother fluorescence-based detection methods can be performed inreal-time. Fluorescence resonance energy transfer (FRET) is theprinciple behind the use of adjacent hybridization probes (Wittwer, C.T. et al. 1997. BioTechniques 22:130-138), molecular beacons (Tyagi S,and Kramer F. R. 1996. Nature Biotechnology 14:303-308) and scorpions(Whitcomb et al. 1999. Nature Biotechnology 17:804-807). Adjacenthybridization probes are designed to be internal to the amplificationprimers. The 3′ end of one probe is labelled with a donor fluorophorewhile the 5′ end of an adjacent probe is labelled with an acceptorfluorophore. When the two probes are specifically hybridized in closedproximity (spaced by 1 to 5 nucleotides) the donor fluorophore which hasbeen excited by an external light source emits light that is absorbed bya second acceptor that emit more fluorescence and yields a FRET signal.Molecular beacons possess a stem-and-loop structure where the loop isthe probe and at the bottom of the stem a fluorescent moiety is at oneend while a quenching moiety is at the other end. The beacons undergo afluorogenic conformational change when they hybridize to their targetshence separating the fluorochrome from its quencher. The FRET principleis also used in an air thermal cycler with a built-in fluorometer(Wittwer, C. T. et al. 1997. BioTechniques 22:130-138). Theamplification and detection are extremely rapid as reactions areperformed in capillaries: it takes only 18 min to complete 45 cycles.Those techniques are suitable especially in the case where few pathogensare searched for. Boehringer-Roche Inc. sells the LightCycler™, andCepheid makes the SmartCycler. These two apparatus are capable of rapidcycle PCR combined with fluorescent SYBR® Green I or FRET detection. Werecently demonstrated in our laboratory, real-time detection of 10 CFUin less than 40 minutes using adjacent hybridization probes on theLightCycler™. Methods based on the detection of fluorescence areparticularly promising for utilization in routine diagnosis as they arevery rapid, quantitative and can be automated.

Microbial pathogens detection and identification may also be performedby solid support or liquid hybridization using species-specific internalDNA probes hybridizing to an amplification product. Such probes may begenerated from any sequence from our repertory and designed tospecifically hybridize to DNA amplification products which are objectsof the present invention. Alternatively, the internal probes for speciesor genus or family or group detection and identification may be derivedfrom the amplicons produced by a universal, family-, group-, genus- orspecies-specific amplification assay(s). The oligonucleotide probes maybe labeled with biotin or with digoxigenin or with any other reportermolecule (for more details see below the section on hybrid capture).Hybrization on a solid support is amendable to miniaturization.

At present the oligonucleotide nucleic acid microarray technology isappealing. Currently, available low to medium density arrays (Heller etal., An integrated microelectronics hybridization system for genomicresearch and diagnostic applications. In: Harrison, D. J., and van denBerg, A., 1998, Micro total analysis systems '98, Kluwer AcademicPublisher, Dordrecht.) could specifically capture fluorescent-labelledamplicons. Detection methods for hybridization are not limited tofluorescence; potentiometry, colorimetry and plasmon resonance are someexamples of alternative detection methods. In addition to detection byhybridization, nucleic acid microarrays could be used to perform rapidsequencing by hybridization. Mass spectrometry could also be applicablefor rapid identification of the amplicon or even for sequencing of theamplification products (Chiu and Cantor, 1999, Clinical Chemistry45:1578; Berkenkamp et al., 1998, Science 281:260).

For the future of our assay format, we also consider the major challengeof molecular diagnostics tools, i.e.: integration of the major stepsincluding sample preparation, genetic amplification, detection, dataanalysis and presentation (Anderson et al., Advances in integratedgenetic analysis. In: Harrison, D. J., and van den Berg, A., 1998, Micrototal analysis systems '98, Kluwer Academic Publisher, Dordrecht.).

To ensure PCR efficiency, glycerol, dimethyl sulfoxide (DMSO) or otherrelated solvents can be used to increase the sensitivity of the PCR andto overcome problems associated with the amplification of a target DNAhaving a high GC content or forming strong secondary structures(Dieffenbach and Dveksler, 1995, PCR Primer: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Plainview, N.Y.). The concentrationranges for glycerol and DMSO are 5-15% (v/v) and 3-10% (v/v),respectively. For the PCR reaction mixture, the concentration ranges forthe amplification primers and MgCl₂ are 0.1-1.5 μM and 1.0-10.0 mM,respectively. Modifications of the standard PCR protocol using externaland nested primers (i.e. nested PCR) or using more than one primer pair(i.e. multiplex PCR) may also be used (Persing et al., 1993, DiagnosticMolecular Microbiology: Principles and Applications, American Societyfor Microbiology, Washington, D.C.). For more details about the PCRprotocols and amplicon detection methods, see Examples.

Hybrid Capture and Chemiluminescence Detection of Amplification Products

Hybridization and detection of amplicons by chemiluminescence wereadapted from Nikiforov et al. (1994, PCR Methods and Applications3:285-291 and 1995, Anal. Biochem. 227:201-209) and from the DIG™ systemprotocol of Boehringer Mannheim. Briefly, 50 μl of a 25 picomolessolution of capture probe diluted in EDC{1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride} areimmobilized in each well of 96-wells plates (Microlite™ 2, Dynex) byincubation overnight at room temperature. The next day, the plates areincubated with a solution of 1% BSA diluted into TNTw (10 mM Tris-HCl,pH 7.5; 150 mM NaCl; 0.05% Tween™ 20) for 1 hour at 37° C. The platesare then washed on a Wellwash Ascent™ (Labsystems) with TNTw followed byWashing Buffer (100 mM maleic acid pH7.5; 150 mM NaCl; 0.3% Tween™ 20).

The amplicons were labelled with DIG-11-dUTP during PCR using the PCRDIG Labelling Mix from Boehringer Mannheim according to themanufacturer's instructions. Hybridization of the amplicons to thecapture probes is performed in triplicate at stringent temperature(generally, probes are designed to allow hybrization at 55° C., thestringent temperature) for 30 minutes in 1.5 M NaCl; 10 mM EDTA. It isfollowed by two washes in 2×SSC; 0.1% SDS, then by four washes in0.1×SSC; 0.1% SDS at the stringent temperature (55° C.). Detection with1,2 dioxetane chemiluminescent alkaline phosphatase substrates likeCSPD® (Tropix Inc.) is performed according to the manufacturer'sinstructions but with shorter incubations times and a different antibodyconcentration. The plates are agitated at each step, the blockingincubation is performed for only 5 minutes, the anti-DIG-AP1 is used ata 1:1000 dilution, the incubation with antibody lasts 15 minutes, theplates are washed twice for only 5 minutes. Finally, after a 2 minutesincubation into the detection buffer, the plates are incubated 5 minuteswith CSPD® at room temperature followed by a 10 minutes incubation at37° C. without agitation. Luminous signal detection is performed on aDynex Microtiter Plate Luminometer using RLU (Relative Light Units).

Specificity, Ubiquity and Sensitivity Tests for Oligonucleotide Primersand Probes

The specificity of oligonucleotide primers and probes was tested byamplification of DNA or by hybridization with bacterial or fungal orparasitical species selected from a panel comprising closely relatedspecies and species sharing the same anatomo-pathological site (seeAnnexes and Examples). All of the bacterial, fungal and parasiticalspecies tested were likely to be pathogens associated with infections orpotential contaminants which can be isolated from clinical specimens.Each target DNA could be released from microbial cells using standardchemical and/or physical treatments to lyse the cells (Sambrook et al.,1989, Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.) or alternatively,genomic DNA purified with the GNOME™ DNA kit (Bio101, Vista, Calif.) wasused. Subsequently, the DNA was subjected to amplification with theprimer pairs. Specific primers or probes amplified only the targetmicrobial species, genus, family or group.

Oligonucleotides primers found to amplify specifically the targetspecies, genus, family or group were subsequently tested for theirubiquity by amplification (i.e. ubiquitous primers amplified efficientlymost or all isolates of the target species or genus or family or group).Finally, the sensitivity of the primers or probes was determined byusing 10-fold or 2-fold dilutions of purified genomic DNA from thetargeted microorganism. For most assays, sensitivity levels in the rangeof 1-100 copies were obtained. The specificity, ubiquity and sensitivityof the PCR assays using the selected amplification primer pairs weretested either directly from cultures of microbial species or frompurified microbial genomic DNA.

Probes were tested in hybrid capture assays as described above. Anoligonucleotide probe was considered specific only when it hybridizedsolely to DNA from the species or genus or family or group from which itwas selected. Oligonucleotide probes found to be specific weresubsequently tested for their ubiquity (i.e. ubiquitous probes detectedefficiently most or all isolates of the target species or genus orfamily or group) by hybridization to microbial DNAs from differentclinical isolates of the species or genus or family or group of interestincluding ATCC reference strains. Similarly, oligonucleotide primers andprobes could be derived from antimicrobial agents resistance or toxingenes which are objects of the present invention.

Reference Strains

The reference strains used to build proprietary tuf, atpD and recAsequence data subrepertories, as well as to test the amplification andhybridization assays were obtained from (i) the American Type CultureCollection (ATCC), (ii) the Laboratoire de santé publique du Québec(LSPQ), (iii) the Centers for Disease Control and Prevention (CDC), (iv)the National Culture Type Collection (NCTC) and (v) several otherreference laboratories throughout the world. The identity of ourreference strains was confirmed by phenotypic testing and reconfirmed byanalysis of tuf, atpD and recA sequences (see Example 13).

Antimicrobial Agents Resistance Genes

Antimicrobial resistance complicates treatment and often leads totherapeutic failures. Furthermore, overuse of antibiotics inevitablyleads to the emergence of microbial resistance. Our goal is to provideclinicians, in approximately one hour, the needed information toprescribe optimal treatments. Besides the rapid identification ofnegative clinical specimens with DNA-based tests for universal algal,archaeal, bacterial, fungal or parasitical detection and theidentification of the presence of a specific pathogen in the positivespecimens with species- and/or genus- and/or family- and/orgroup-specific DNA-based tests, clinicians also need timely informationabout the ability of the microbial pathogen to resist antibiotictreatments. We feel that the most efficient strategy to evaluate rapidlymicrobial resistance to antimicrobials is to detect directly from theclinical specimens the most common and clinically importantantimicrobial agents resistance genes (i.e. DNA-based tests for thespecific detection of antimicrobial agents resistance genes). Since thesequence from the most important and common antimicrobial agentsresistance genes are available from public databases, our strategy is touse the sequence from a portion or from the entire resistance gene todesign specific oligonucleotide primers or probes which will be used asa basis for the development of sensitive and rapid DNA-based tests. Thelist of each of the antimicrobial agents resistance genes selected onthe basis of their clinical relevance (i.e. high incidence andimportance) is given in Table 5; descriptions of the designedamplification primers and internal probes are given in Tables 72-75, 77,84, and 88-90. Our approach is unique because the antimicrobial agentsresistance genes detection and the microbial detection andidentification can be performed simultaneously, or independently, orsequentially in multiplex or parallel or sequential assays under uniformPCR amplification conditions. These amplifications can also be doneseparately.

Toxin Genes

Toxin identification is often very important to prescribe optimaltreatments. Besides the rapid identification of negative clinicalspecimens with DNA-based tests for universal bacterial detection and theidentification of the presence of a specific pathogen in the positivespecimens with species- and/or genus- and/or family- and/orgroup-specific DNA-based tests, clinicians sometimes need timelyinformation about the ability of certain bacterial pathogens to producetoxins. Since the sequence from the most important and common bacterialtoxin genes are available from public databases, our strategy is to usethe sequence from a portion or from the entire toxin gene to designspecific oligonucleotide primers or probes which will be used as a basisfor the development of sensitive and rapid DNA-based tests. The list ofeach of the bacterial toxin genes selected on the basis of theirclinical relevance (i.e. high incidence and importance) is given inTable 6; descriptions of the designed amplification primers and internalprobes are given in Tables 60, 70 and 71. Our approach is unique becausethe toxin genes detection and the bacterial detection and identificationcan be performed simultaneously, or independently, or sequentially, inmultiplex or parallel or sequential assays under uniform PCRamplification conditions. These amplifications can also be doneseparately.

Universal Bacterial Detection

In the routine microbiology laboratory, a high percentage of clinicalspecimens sent for bacterial identification are negative by culture.Testing clinical samples with universal amplification primers oruniversal probes to detect the presence of bacteria prior to specificidentification and screening out the numerous negative specimens is thususeful as it reduces costs and may rapidly orient the clinicalmanagement of the patients. Several amplification primers and probeswere therefore synthesized from highly conserved portions of bacterialsequences from the tuf, atpD and recA nucleic acids and/or sequences.The universal primers selection was based on a multiple sequencealignment constructed with sequences from our repertory.

All computer analysis of amino acid and nucleotide sequences wereperformed by using the GCG programs. Subsequently, optimal PCR primersfor the universal amplification of bacteria were selected with the helpof the Oligo™ program. The selected primers are degenerated at severalnucleotide positions and contain several inosines in order to allow theamplification of all clinically relevant bacterial species. Inosine is anucleotide analog able to specifically bind to any of the fournucleotides A, C, G or T. Degenerated oligonucleotides consist of anoligonucleotide mix having two or more of the four nucleotides A, C, Gor T at the site of mismatches. The inclusion of inosine and/or of baseambiguities in the amplification primers allow mismatch tolerancethereby permitting the amplification of a wider array of targetnucleotide sequences (Dieffenbach and Dveksler, 1995 PCR Primer: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Plainview,N.Y.).

The amplification conditions with the universal primers are very similarto those used for the species- and genus-specific amplification assaysexcept that the annealing temperature is slightly lower. The originaluniversal PCR assay described in our assigned WO98/20157 (SEQ ID NOs.23-24 of the latter application) was specific and nearly ubiquitous forthe detection of bacteria. The specificity for bacteria was verified byamplifying genomic DNA isolated from the 12 fungal species as well asgenomic DNA from Leishmania donovani, Saccharomyces cerevisiae and humanlymphocytes. None of the above eukaryotic DNA preparations could beamplified by the universal assay, thereby suggesting that this test isspecific for bacteria. The ubiquity of the universal assay was verifiedby amplifying genomic DNAs from 116 reference strains which represent 95of the most clinically relevant bacterial species. These species havebeen selected from the bacterial species listed in Table 4. We foundthat at least 104 of these strains could be amplified. However, theassay could be improved since bacterial species which could not beamplified with the original tuf nucleic acids and/or sequences-basedassay included species belonging to the following genera:Corynebacterium (11 species) and Stenotrophomonas (1 species).Sequencing of the tuf genes from these bacterial species and others hasbeen performed in the scope of the present invention in order to improvethe universal assay. This sequencing data has been used to select newuniversal primers which may be more ubiquitous and more sensitive. Also,we improved our primer and probes design strategy by taking intoconsideration the phylogeny observed in analysing our repertory of tuf,atpD and recA sequences. Data from each of the 3 main subrepertories(tuf, atpD and recA) was subjected to a basic phylogenic analysis usingthe Pileup command from version 10 of the GCG package (Genetics ComputerGroup, inc.). This analysis indicated the main branches or phylareflecting the relationships between sequences. Instead of trying todesign primers or probes able to hybridize to all phyla, we designedprimers or probes able to hybridize to the main phyla while trying touse the largest phylum possible. This strategy should allow lessdegenerated primers hence improving sensitivity and by combining primersin a mutiplex assay, improve ubiquity. Universal primers SEQ ID NOs.643-645 based on tuf sequences have been designed to amplify mostpathogenic bacteria except Actinomyceteae, Clostridiaceae and theCytophaga, Flexibacter and Bacteroides phylum (pathogenic bacteria ofthis phylum include mostly Bacteroides, Porphyromonas and Prevotellaspecies). Primers to fill these gaps have been designed forActinomyceteae (SEQ ID NOs. 646-648), Clostridiaceae (SEQ ID NOs.796-797, 808-811), and the Cytophaga, Flexibacter and Bacteroides phylum(SEQ ID NOs. 649-651), also derived from tuf nucleic acids and/orsequences. These primers sets could be used alone or in conjuction torender the universal assay more ubiquitous.

Universal primers derived from atpD sequences include SEQ ID NOs.562-565. Combination of these primers does not amplify human DNA butshould amplify almost all pathogenic bacterial species exceptproteobacteria belonging to the epsilon subdivision (Campylobacter andHelicobacter), the bacteria from the Cytophaga, Flexibacter andBacteroides group and some actinomycetes and corynebacteria. Byanalysing atpD sequences from the latter species, primers and probes tospecifically fill these gaps could be designed and used in conjuctionwith primers SEQ ID NOs. 562-565, also derived from atpD nucleic acidsand/or sequences.

In addition, universality of the assay could be expanded by mixing atpDsequences-derived primers with tuf sequences-derived primers.Ultimately, even recA sequences-derived primers could be added to fillsome gaps in the universal assay.

It is important to note that the 95 bacterial species selected to testthe ubiquity of the universal assay include all of the most clinicallyrelevant bacterial species associated with a variety of human infectionsacquired in the community or in hospitals (nosocomial infections). Themost clinically important bacterial and fungal pathogens are listed inTables 1 and 2.

Amino Acid Sequences Derived from Tuf, atpD and recA Nucleic Acidsand/or Sequences

The amino acid sequences translated from the repertory of tuf, atpD andrecA nucleic acids and/or sequences are also an object of the presentinvention. The amino acid sequence data will be particularly useful forhomology modeling of three-dimensional (3D) structure of the elongationfactor Tu, elongation factor G, elongation factor 1a, ATPase subunitbeta and RecA recombinase. For all these proteins, at least onestructure model has been published using X-ray diffraction data fromcrystals. Based on those structural informations it is possible to usecomputer software to build 3D model structures for any other proteinhaving peptide sequence homologies with the known structure (Greer,1991, Methods in Enzymology, 202:239-252; Taylor, 1994, TrendsBiotechnol., 12(5):154-158; Sali, 1995, Curr. Opin. Biotechnol.6:437-451; Sanchez and Sali, 1997, Curr. Opin. Struct. Biol. 7:206-214;Fischer and Eisenberg, 1999, Curr. Opin. Struct. Biol. 9:208-211; Guexet al., 1999, Trends Biochem. Sci. 24: 364-367). Model structures oftarget proteins are used for the design or to predict the behavior ofligands and inhibitors such as antibiotics. Since EF-Tu and EF-G arealready known as antibiotic targets (see above) and since the betasubunit of ATPase and RecA recombinase are essential to the survival ofthe microbial cells in natural conditions of infection, all fourproteins could be considered antibiotic targets. Sequence data,especially the new data generated by us could be very useful to assistthe creation of new antibiotic molecules with desired spectrum ofactivity. In addition, model structures could be used to improve proteinfunction for commercial purposes such as improving antibiotic productionby microbial strains or increasing biomass.

The following detailed embodiments and appended drawings are provided asillustrative examples of his invention, with no intention to limit thescope thereof.

Examples and Annexes

For sake of clarity, here is a list of Examples and Annexes:

Example 1: Sequencing of bacterial atpD (F-type and V-type) genefragments.

Example 2: Sequencing of eukaryotic atpD (F-type and V-type) genefragments.

Example 3: Sequencing of eukaryotic tuf (EF-1) gene fragments.

Example 4: Sequencing of eukaryotic tuf (organelle origin, M) genefragments.

Example 5: Specific detection and identification of Streptococcusagalactiae using tuf sequences.

Example 6: Specific detection and identification of Streptococcusagalactiae using atpD sequences.

Example 7: Development of a PCR assay for detection and identificationof staphylococci at genus and species levels.

Example 8: Differentiating between the two closely related yeast speciesCandida albicans and Candida dubliniensis.

Example 9: Specific detection and identification of Entamoebahistolytica.

Example 10: Sensitive detection and identification of Chlamydiatrachomatis.

Example 11: Genus-specific detection and identification of enterococci.

Example 12: Detection and identification of the major bacterialplatelets contaminants using tuf sequences with a multiplex PCR test.

Example 13: The resolving power of the tuf and atpD sequences databasesis comparable to the biochemical methods for bacterial identification.

Example 14: Detection of group B streptococci from clinical specimens.

Example 15: Simultaneous detection and identification of Streptococcuspyogenes and its pyrogenic exotoxin A.

Example 16: Real-time detection and identification of Shigatoxin-producing bacteria.

Example 17: Development of a PCR assay for the detection andidentification of staphylococci at genus and species levels and itsassociated mecA gene.

Example 18: Sequencing of pbp1a, pbp2b and pbp2x genes of Streptoccoccuspneumoniae.

Example 19: Sequencing of hexA genes of Streptococcus species.

Example 20: Development of a multiplex PCR assay for the detection ofStreptococcus pneumoniae and its penicillin resistance genes.

Example 21: Sequencing of the vancomycin resistance vanA, vanC1, vanC2and vanC3 genes.

Example 22: Development of a PCR assay for the detection andidentification of enterococci at genus and species levels and itsassociated resistance genes vanA and vanB.

Example 23: Development of a multiplex PCR assay for detection andidentification of vancomycin-resistant Enterococcus faecalis,Enterococcus faecium, Enterococcus gallinarum, Enterococcuscasseliflavus, and Enterococcus flavescens.

Example 24: Universal amplification involving the EF-G (fusA)subdivision of tuf sequences.

Example 25: DNA fragment isolation from Staphylococcus saprophyticus byarbitrarily primed PCR.

Example 26: Sequencing of prokaryotic tuf gene fragments.

Example 27: Sequencing of procaryotic recA gene fragments.

Example 28: Specific detection and identification of Escherichiacoli/Shigella sp. using tuf sequences.

Example 29: Specific detection and identification of Klebsiellapneumoniae using atpD sequences.

Example 30: Specific detection and identification of Acinetobacterbaumanii using tuf sequences.

Example 31: Specific detection and identification of Neisseriagonorrhoeae using tuf sequences.

Example 32: Sequencing of bacterial gyrA and parC gene fragments.

Example 33: Development of a PCR assay for the specific detection andidentification of Staphylococcus aureus and its quinolone resistancegenes gyrA and parC.

Example 34: Development of a PCR assay for the detection andidentification of Klebsiella pneumoniae and its quinolone resistancegenes gyrA and parC.

Example 35: Development of a PCR assay for the detection andidentification of Streptococcus pneumoniae and its quinolone resistancegenes gyrA and parC.

Example 36: Detection of extended-spectrum TEM-type β-lactamases inEscherichia coli.

Example 37: Detection of extended-spectrum SHV-type β-lactamases inKlebsiella pneumoniae.

Example 38: Development of a PCR assay for the detection andidentification of Neisseria gonorrhoeae and its associated tetracyclineresistance gene tetM.

Example 39: Development of a PCR assay for the detection andidentification of Shigella sp. and their associated trimethoprimresistance gene dhfr1a.

Example 40: Development of a PCR assay for the detection andidentification of Acinetobacter baumanii and its associatedaminoglycoside resistance gene aph(3′)-VIa.

Example 41: Specific detection and identification of Bacteroidesfragilis using atpD (V-type) sequences.

Example 42: Evidence for horizontal gene transfer in the evolution ofthe elongation factor Tu in Enterococci.

Example 43: Elongation factor Tu (tuf) and the F-ATPase beta-subunit(atpD) as phylogenetic tools for species of the familyEnterobacteriaceae.

Example 44: Testing new pairs of PCR primers selected from twospecies-specific genomic DNA fragments which are objects of U.S. Pat.No. 6,001,564.

Example 45: Testing modified versions of PCR primers derived from thesequence of several primers which are objects of U.S. Pat. No.6,001,564.

The various Annexes show the strategies used for the selection of avariety of DNA amplification primers, nucleic acid hybridization probesand molecular beacon internal probes:

-   -   (i) Table 39 shows the amplification primers used for nucleic        acid amplification from tuf sequences.    -   (ii) Table 40 shows the amplification primers used for nucleic        acid amplification from atpD sequences.    -   (iii) Table 41 shows the internal hybridization probes for        detection of tuf sequences.    -   (iv) Table 42 illustrates the strategy used for the selection of        the amplification primers specific for atpD sequences of the        F-type.    -   (v) Table 43 illustrates the strategy used for the selection of        the amplification primers specific for atpD sequences of the        V-type.    -   (vi) Table 44 illustrates the strategy used for the selection of        the amplification primers specific for the tuf sequences of        organelle lineage (M, the letter M is used to indicate that in        most cases, the organelle is the mitochondria).    -   (vii) Table 45 illustrates the strategy used for the selection        of the amplification primers specific for the tuf sequences of        eukaryotes (EF-1).    -   (viii) Table 46 illustrates the strategy for the selection of        Streptococcus agalactiae-specific amplification primers from tuf        sequences.    -   (ix) Table 47 illustrates the strategy for the selection of        Streptococcus agalactiae-specific hybridization probes from tuf        sequences.    -   (x) Table 48 illustrates the strategy for the selection of        Streptococcus agalactiae-specific amplification primers from        atpD sequences.    -   (xi) Table 49 illustrates the strategy for the selection from        tuf sequences of Candida albicans/dubliniensis-specific        amplification primers, Candida albicans-specific hybridization        probe and Candida dubliniensis-specific hybridization probe.    -   (xii) Table 50 illustrates the strategy for the selection of        Staphylococcus-specific amplification primers from tuf        sequences.    -   (xiii) Table 51 illustrates the strategy for the selection of        the Staphylococcus-specific hybridization probe from tuf        sequences.    -   (xiv) Table 52 illustrates the strategy for the selection of        Staphylococcus saprophyticus-specific and Staphylococcus        haemolyticus-specific hybridization probes from tuf sequences.    -   (xv) Table 53 illustrates the strategy for the selection of        Staphylococcus aureus-specific and Staphylococcus        epidermidis-specific hybridization probes from tuf sequences.    -   (xvi) Table 54 illustrates the strategy for the selection of the        Staphylococcus hominis-specific hybridization probe from tuf        sequences.    -   (xvii) Table 55 illustrates the strategy for the selection of        the Enterococcus-specific amplification primers from tuf        sequences.    -   (xviii) Table 56 illustrates the strategy for the selection of        the Enterococcus faecalis-specific hybridization probe, of the        Enterococcus faecium-specific hybridization probe and of the        Enterococcus casseliflavus-flavescens-gallinarum group-specific        hybridization probe from tuf sequences.    -   (xix) Table 57 illustrates the strategy for the selection of        primers from tuf sequences for the identification of platelets        contaminants.    -   (xx) Table 58 illustrates the strategy for the selection of the        universal amplification primers from atpD sequences.    -   (xxi) Table 59 shows the amplification primers used for nucleic        acid amplification from recA sequences.    -   (xxii) Table 60 shows the specific and ubiquitous primers for        nucleic acid amplification from speA sequences.    -   (xxiii) Table 61 illustrates the first strategy for the        selection of Streptococcus pyogenes-specific amplification        primers from speA sequences.    -   (xxiv) Table 62 illustrates the second strategy for the        selection of Streptococcus pyogenes-specific amplification        primers from speA sequences.    -   (xxv) Table 63 illustrates the strategy for the selection of        Streptococcus pyogenes-specific amplification primers from tuf        sequences.    -   (xxvi) Table 64 illustrates the strategy for the selection of        stx₁-specific amplification primers and hybridization probe.    -   (xxvii) Table 65 illustrates the strategy for the selection of        stx₂-specific amplification primers and hybridization probe.    -   (xxviii) Table 66 illustrates the strategy for the selection of        vanA-specific amplification primers from van sequences.    -   (xxix) Table 67 illustrates the strategy for the selection of        vanB-specific amplification primers from van sequences.    -   (xxx) Table 68 illustrates the strategy for the selection of        vanC-specific amplification primers from vanC sequences.    -   (xxxi) Table 69 illustrates the strategy for the selection of        Streptococcus pneumoniae-specific amplification primers and        hybridization probes from pbp1a sequences.    -   (xxxii) Table 70 shows the specific and ubiquitous primers for        nucleic acid amplification from toxin gene sequences.    -   (xxxiii) Table 71 shows the molecular beacon internal        hybridization probes for specific detection of toxin sequences.    -   (xxxiv) Table 72 shows the specific and ubiquitous primers for        nucleic acid amplification from van sequences.    -   (xxxv) Table 73 shows the internal hybridization probes for        specific detection of van sequences.    -   (xxxvi) Table 74 shows the specific and ubiquitous primers for        nucleic acid amplification from pbp sequences.    -   (xxxvii) Table 75 shows the internal hybridization probes for        specific detection of pbp sequences.    -   (xxxviii) Table 76 illustrates the strategy for the selection of        vanAB-specific amplification primers and vanA- and vanB-specific        hybridization probes from van sequences.    -   (xxxix) Table 77 shows the internal hybridization probe for        specific detection of mecA.    -   (xl) Table 78 shows the specific and ubiquitous primers for        nucleic acid amplification from hexA sequences.    -   (xli) Table 79 shows the internal hybridization probe for        specific detection of hexA.    -   (xlii) Table 80 illustrates the strategy for the selection of        Streptococcus pneumoniae species-specific amplification primers        and hybridization probe from hexA sequences.    -   (xliii) Table 81 shows the specific and ubiquitous primers for        nucleic acid amplification from pcp sequences.    -   (xliv) Table 82 shows specific and ubiquitous primers for        nucleic acid amplification of S. saprophyticus sequences of        unknown coding potential.    -   (xlv) Table 83 shows the molecular beacon internal hybridization        probes for specific detection of antimicrobial agents resistance        gene sequences.    -   (xlvi) Table 84 shows the molecular beacon internal        hybridization probe for specific detection of S. aureus gene        sequences of unknown coding potential.    -   (xlvii) Table 85 shows the molecular beacon hybridization        internal probe for specific detection of tuf sequences.    -   (xlviii) Table 86 shows the molecular beacon internal        hybridization probes for specific detection of ddI and mtl        sequences.    -   (xlix) Table 87 shows the internal hybridization probe for        specific detection of S. aureus sequences of unknown coding        potential.    -   (l) Table 88 shows the amplification primers used for nucleic        acid amplification from antimicrobial agents resistance genes        sequences.    -   (li) Table 89 shows the internal hybridization probes for        specific detection of antimicrobial agents resistance genes        sequences.    -   (lii) Table 90 shows the molecular beacon internal hybridization        probes for specific detection of atpD sequences.    -   (liii) Annex Table 91 shows the internal hybridization probes        for specific detection of atpD sequences.    -   (liv) Table 92 shows the internal hybridization probes for        specific detection of ddI and mtl sequences.

As shown in these Annexes, the selected amplification primers maycontain inosines and/or base ambiguities. Inosine is a nucleotide analogable to specifically bind to any of the four nucleotides A, C, G or T.Alternatively, degenerated oligonucleotides which consist of anoligonucleotide mix having two or more of the four nucleotides A, C, Gor T at the site of mismatches were used. The inclusion of inosineand/or of degeneracies in the amplification primers allows mismatchtolerance thereby permitting the amplification of a wider array oftarget nucleotide sequences (Dieffenbach and Dveksler, 1995 PCR Primer:A Laboratory Manual, Cold Spring Harbor Laboratory Press, Plainview,N.Y.).

EXAMPLES Example 1 Sequencing of Bacterial atpD (F-Type and V-Type) GeneFragments

As shown in Table 42, the comparison of publicly available atpD (F-type)sequences from a variety of bacterial species revealed conserved regionsallowing the design of PCR primers able to amplify atpD sequences(F-type) from a wide range of bacterial species. Using primers pairs SEQID NOs. 566 and 567, 566 and 814, 568 and 567, 570 and 567, 572 and 567,569 and 567, 571 and 567, 700 and 567, it was possible to amplify andsequence atpD sequences SEQ ID NOs. 242-270, 272-398, 673-674, 737-767,866-867, 942-955, 1245-1254, 1256-1265, 1527, 1576, 1577, 1600-1604,1640-1646, 1649, 1652, 1655, 1657, 1659-1660, 1671, 1844-1845, and1849-1865.

Similarly, Table 43 shows the strategy to design the PCR primers able toamplify atpD sequences of the V-type from a wide range of archaeal andbacterial species. Using primers SEQ ID NOs. 681-683, it was possible toamplify and sequence atpD sequences SEQ ID NOs. 827-832, 929-931, 958and 966. As the gene was difficult to amplify for several species,additional amplification primers were designed inside the originalamplicon (SEQ ID NOs. 1203-1207) in order to obtain sequence informationfor these species. Other primers (SEQ ID NO. 1212, 1213, 2282-2285) werealso designed to amplify regions of the atpD gene (V-type) inarchaebacteria.

Example 2 Sequencing of Eukaryotic atpD (F-Type and V-Type) GeneFragments

The comparison of publicly available atpD (F-type) sequences from avariety of fungal and parasitical species revealed conserved regionsallowing the design of PCR primers able to amplify atpD sequences from awide range of fungal and parasitical species. Using primers pairs SEQ IDNOs. 568 and 573, 574 and 573, 574 and 708, and 566 and 567, it waspossible to amplify and sequence atpD sequences SEQ ID NOs. 458-497,530-538, 663, 667, 676, 678-680, 768-778, 856-862, 889-896, 941,1638-1639, 1647, 1650-1651, 1653-1654, 1656, 1658, 1684, 1846-1848, and2189-2192.

In the same manner, the primers described in Table 43 (SEQ ID NOs.681-683) could amplify the atpD (V-type) gene from various fungal andparasitical species. This strategy allowed to obtain SEQ ID NOs.834-839, 956-957, and 959-965.

Example 3 Sequencing of Eukaryotic tuf (EF-1) Gene Fragments

As shown in Table 45, the comparison of publicly available tuf (EF-1)sequences from a variety of fungal and parasitical species revealedconserved regions allowing the design of PCR primers able to amplify tufsequences from a wide range of fungal and parasitical species. Usingprimers pairs SEQ ID NOs. 558 and 559, 813 and 559, 558 and 815, 560 and559, 653 and 559, 558 and 655, and 654 and 559, 1999 and 2000, 2001 and2003, 2002 and 2003, it was possible to amplify and sequence tufsequences SEQ ID NOs. 399-457, 509-529, 622-624, 677, 779-790, 840-842,865, 897-903, 1266-1287, 1561-1571 and 1685.

Example 4 Sequencing of Eukaryotic tuf (Organelle Origin, M) GeneFragments

As shown in Table 44, the comparison of publicly available tuf(organelle origin, M) sequences from a variety of fungal and parasiticalorganelles revealed conserved regions allowing the design of PCR primersable to amplify tuf sequences of several organelles belonging to a widerange fungal and parasitical species. Using primers pairs SEQ ID NOs.664 and 652, 664 and 561, 911 and 914, 912 and 914, 913 and 915, 916 and561, 664 and 917, it was possible to amplify and sequence tuf sequencesSEQ ID NOs. 498-508, 791-792, 843-855, 904-910, 1664, 1666-1667,1669-1670, 1673-1683, 1686-1689, 1874-1876, 1879, 1956-1960, and2193-2199.

Example 5 Specific Detection and Identification of Streptococcusagalactiae Using tuf Sequences

As shown in Table 46, the comparison of tuf sequences from a variety ofbacterial species allowed the selection of PCR primers specific for S.agalactiae. The strategy used to design the PCR primers was based on theanalysis of a multiple sequence alignment of various tuf sequences. Themultiple sequence alignment includes the tuf sequences of four bacterialstrains from the target species as well as tuf sequences from otherspecies and bacterial genera, especially representatives of closelyrelated species. A careful analysis of this alignment allowed theselection of oligonucleotide sequences which are conserved within thetarget species but which discriminate sequences from other species andgenera, especially from the closely related species, thereby permittingthe species-specific, ubiquitous and sensitive detection andidentification of the target bacterial species.

The chosen primer pair, oligos SEQ ID NO. 549 and SEQ ID NO. 550, givesan amplification product of 252 bp. Standard PCR was carried out using0.4 μM of each primer, 2.5 mM MgCl₂, BSA 0.05 mM, 1× Taq Buffer(Promega), dNTP 0.2 mM (Pharmacia), 0.5 U Taq DNA polymerase (Promega)coupled with TaqStart™ antibody (Clontech Laboratories Inc., Palo Alto),1 μl of genomic DNA sample in a final volume of 20 μl using a PTC-200thermocycler (MJ Research Inc.). The optimal cycling conditions formaximum sensitivity and specificity were 3 minutes at 95° C. for initialdenaturation, then forty cycles of two steps consisting of 1 second at95° C. and 30 seconds at 62° C., followed by terminal extension at 72°C. for 2 minutes. Detection of the PCR products was made byelectrophoresis in agarose gels (2%) containing 0.25 μg/ml of ethidiumbromide.

Specificity of the assay was tested by adding into the PCR reactions,0.1 ng of genomic DNA from each of the bacterial species listed in Table8. Efficient amplification was observed only for the 5 S. agalactiaestrains listed. Of the other bacterial species, including 32 speciesrepresentative of the vaginal flora and 27 other streptococcal species,only S. acidominimus yielded amplification. The signal with 0.1 ng of S.acidominimus genomic DNA was weak and the detection limit for thisspecies was 10 pg (corresponding to more than 4000 genome copies) whilethe detection limit for S. agalactiae was 2.5 fg (corresponding to onegenome copy) of genomic DNA.

To increase the specificity of the assay, internal probes were designedfor FRET (Fluorescence Resonance Energy Transfer) detection using theLightCycler™ (Idaho Technology). As illustrated in Table 47, a multiplesequence alignment of streptococcal tuf sequence fragments correspondingto the 252 bp region amplified by primers SEQ ID NO. 549 and SEQ ID NO.550, was used for the design of internal probes TSagHF436 (SEQ ID NO.582) and TSagHF465 (SEQ ID NO. 583). The region of the amplicon selectedfor internal probes contained sequences unique and specific to S.agalactiae. SEQ ID NO. 583, the more specific probe, is labelled withfluorescein in 3′, while SEQ ID NO. 582, the less discriminant probe, islabelled with CY5 in 5′ and blocked in 3′ with a phosphate group.However, since the FRET signal is only emitted if both probes areadjacently hybridized on the same target amplicon, detection is highlyspecific.

Real-time detection of PCR products using the LightCycler™ was carriedout using 0.4 μM of each primer (SEQ ID NO. 549-550), 0.2 μM of eachprobe (SEQ ID NO. 582-583), 2.5 mM MgCl₂, BSA 450 μg/ml, 1×PC2 Buffer(AB Peptides, St-Louis, Mo.), dNTP 0.2 mM (Pharmacia), 0.5 U KlenTaq1™DNA polymerase (AB Peptides) coupled with TaqStart™ antibody (ClontechLaboratories Inc., Palo Alto), 0.7 μl of genomic DNA sample in a finalvolume of 7 μl using a LightCycler thermocycler (Idaho Technology). Theoptimal cycling conditions for maximum sensitivity and specificity were3 minutes at 94° C. for initial denaturation, then forty cycles of threesteps consisting of 0 second (this setting meaning the LightCycler willreach the target temperature and stay at it for its minimal amount oftime) at 94° C., 10 seconds at 64° C., 20 seconds at 72° C.Amplification was monitored during each annealing steps using thefluorescence ratio. The streptococcal species having close sequencehomologies with the tuf sequence of S. agalactiae (S. acidominimus, S.anginosus, S. Bovis, S. dysgalactiae, S. equi, S. ferus, S. gordonii, S.intermedius, S. parasanguis, S. parauberis, S. salivarius, S. sanguis,S. suis) as well as S. agalactiae were tested in the LightCycler with0.07 ng of genomic DNA per reaction. Only S. agalactiae yielded anamplification signal, hence demonstrating that the assay isspecies-specific. With the LightCycler™ assay using the internal FRETprobes, the detection limit for S. agalactiae was 1-2 genome copies ofgenomic DNA.

Example 6 Specific Detection and Identification of Streptococcusagalactiae Using atpD Sequences

As shown in Table 48, the comparison of atpD sequences from a variety ofbacterial species allowed the selection of PCR primers specific for S.agalactiae. The primer design strategy is similar to the strategydescribed in the preceding Example except that atpD sequences were usedin the alignment.

Four primers were selected, ASag42 (SEQ ID NO. 627), ASag52 (SEQ ID NO.628), ASag206 (SEQ ID NO. 625) and ASag371 (SEQ ID NO. 626). Thefollowing combinations of these four primers give four amplicons; SEQ IDNO. 627+SEQ ID NO. 625=190 bp, SEQ ID NO. 628+SEQ ID NO. 625=180 bp, SEQID NO. 627+SEQ ID NO. 626=355 bp, and SEQ ID NO. 628+SEQ ID NO. 626=345bp.

Standard PCR was carried out on PTC-200 thermocyclers (MJ Research Inc)using 0.4 μM of each primers pair, 2.5 mM MgCl₂, BSA 0.05 mM, 1× taqBuffer (Promega), dNTP 0.2 mM (Pharmacia), 0.5 U Taq DNA polymerase(Promega) coupled with TaqStart™ antibody (Clontech Laboratories Inc.,Palo Alto), 1 μl of genomic DNA sample in a final volume of 20 μL. Theoptimal cycling conditions for maximum sensitivity and specificity wereadjusted for each primer pair. Three minutes at 95° C. for initialdenaturation, then forty cycles of two steps consisting of 1 second at95° C. and 30 seconds at the optimal annealing temperature specifiedbelow were followed by terminal extension at 72° C. for 2 minutes.Detection of the PCR products was made by electrophoresis in agarosegels (2%) containing 0.25 μg/ml of ethidium bromide. Since atpDsequences are relatively more specific than tuf sequences, only the mostclosely related species namely, the steptococcal species listed in Table9, were tested.

All four primer pairs only amplified the six S. agalactiae strains. Withan annealing temperature of 63° C., the primer pair SEQ ID NO. 627+SEQID NO. 625 had a sensitivity of 1-5 fg (equivalent to 1-2 genomecopies). At 55° C., the primer pair SEQ ID NO. 628+SEQ ID NO. 625 had asensitivity of 2.5 fg (equivalent to 1 genome copy). At 60° C., theprimer pair SEQ ID NO. 627+SEQ ID NO. 626 had a sensitivity of 10 fg(equivalent to 4 genome copies). At 58° C., the primer pair SEQ ID NO.628+SEQ ID NO. 626 had a sensitivity of 2.5-5 fg (equivalent to 1-2genome copies). This proves that all four primer pairs can detect S.agalactiae with high specificity and sensitivity. Together with Example5, this example demonstrates that both tuf and atpD sequences aresuitable and flexible targets for the identification of microorganismsat the species level. The fact that 4 different primer pairs based onatpD sequences led to efficient and specific amplification of S.agalactiae demonstrates that the challenge is to find target genessuitable for diagnostic purposes, rather than finding primer pairs fromthese target sequences.

Example 7 Development of a PCR Assay for Detection and Identification ofStaphylococci at Genus and Species Levels

Materials and Methods

Bacterial strains. The specificity of the PCR assay was verified byusing a panel of ATCC (America Type Culture Collection) and DSMZ(Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH; GermanCollection of Microorganisms and Cell Cultures) reference strainsconsisting of 33 gram-negative and 47 gram-positive bacterial species(Table 12). In addition, 295 clinical isolates representing 11 differentspecies of staphylococci from the microbiology laboratory of the CentreHospitalier Universitaire de Québec, Pavillon Centre Hospitalier del'Université Laval (CHUL) (Step-Foy, Québec, Canada) were also tested tofurther validate the Staphylococcus-specific PCR assay. These strainswere all identified by using (i) conventional methods or (ii) theautomated MicroScan Autoscan-4 system equipped with the Positive BPCombo Panel Type 6 (Dade Diagnostics, Mississauga, Ontario, Canada).Bacterial strains from frozen stocks kept at −80° C. in brain heartinfusion (BHI) broth containing 10% glycerol were cultured on sheepblood agar or in BHI broth (Quelab Laboratories Inc, Montréal, Québec,Canada).

PCR primers and internal probes. Based on multiple sequence alignments,regions of the tuf gene unique to staphylococci were identified.Staphylococcus-specific PCR primers TStaG422 (SEQ ID NO. 553) andTStaG765 (SEQ ID NO. 575) were derived from these regions (Table 50).These PCR primers are displaced by two nucleotide positions compared tooriginal Staphylococcus-specific PCR primers described in our patentpublication WO98/20157 (SEQ ID NOs. 17 and 20 in the said patentpublication). These modifications were done to ensure specificity andubiquity of the primer pair, in the light of new tuf sequence datarevealed in the present patent application for several additionalstaphylococcal species and strains.

Similarly, sequence alignment analysis were performed to design genusand species-specific internal probes (see Tables 61 to 64). Two internalprobes specific for Staphylococcus (SEQ ID NOs. 605-606), five specificfor S. aureus (SEQ ID NOs. 584-588), five specific for S. epidermidis(SEQ ID NO. 589-593), two specific for S. haemolyticus (SEQ ID NOs.594-595), three specific for S. hominis (SEQ ID NOs. 596-598), fourspecific for S. saprophyticus (SEQ ID NOs. 599-601 and 695), and twospecific for coagulase-negative Staphylococcus species including S.epidermidis, S. hominis, S. saprophyticus, S. auricularis, S. capitis,S. haemolyticus, S. lugdunensis, S. simulans, S. cohnii and S. warneri(SEQ ID NOs. 1175-1176) were designed. The range of mismatches betweenthe Staphylococcus-specific 371-bp amplicon and each of the 20-merspecies-specific internal probes was from 1 to 5, in the middle of theprobe when possible. No mismatches were present in the twoStaphylococcus-specific probes for the 11 species analyzed: S. aureus,S. auricularis, S. capitis, S. cohnii, S. epidermidis, S. haemolyticus,S. hominis, S. lugdunensis, S. saprophyticus, S. simulans and S.warneri. In order to verify the intra-specific sequence conservation ofthe nucleotide sequence, sequences were obtained for the 371-bp ampliconfrom five unrelated ATCC and clinical strains for each of the species S.aureus, S. epidermidis, S. haemolyticus, S. hominis and S.saprophyticus. The Oligo™ (version 5.0) primer analysis software(National Biosciences, Plymouth, Minn.) was used to confirm the absenceof self-complementary regions within and between the primers or probes.When required, the primers contained inosines or degenerated nucleotidesat one or more variable positions. Oligonucleotide primers and probeswere synthesized on a model 394 DNA synthesizer (Applied Biosystems,Mississauga, Ontario, Canada). Detection of the hybridization wasperformed with the DIG-labeled dUTP incorporated during amplificationwith the Staphylococcus-specific PCR assay, and the hybridization signalwas detected with a luminometer (Dynex Technologies) as described abovein the section on luminescent detection of amplification products.Tables 61 to 64 illustrate the strategy for the selection of severalinternal probes.

PCR amplification. For all bacterial species, amplification wasperformed from purified genomic DNA or from a bacterial suspension whoseturbidity was adjusted to that of a 0.5 McFarland standard, whichcorresponds to approximately 1.5×10⁸ bacteria per ml. One nanogram ofgenomic DNA or 1 □l of the standardized bacterial suspension wastransferred directly to a 19 □l PCR mixture. Each PCR reaction contained50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM MgCl₂,0.20M (each) of the two Staphylococcus genus-specific primers (SEQ IDNOs. 553 and 575), 2000M (each) of the four deoxynucleosidetriphosphates (Pharmacia Biotech), 3.3 □g/□l bovine serum albumin (BSA)(Sigma-Aldrich Canada Ltd, Oakville, Ontario, Canada), and 0.5 U Taqpolymerase (Promega) coupled with TaqStart™ Antibody (Clontech). The PCRamplification was performed as follows: 3 min. at 94° C. for initialdenaturation, then forty cycles of two steps consisting of 1 second at95° C. and 30 seconds at 55° C., plus a terminal extension at 72° C. for2 minutes. Detection of the PCR products was made by electrophoresis inagarose gels (2%) containing 0.25 μg/ml of ethidium bromide.Visualization of the PCR products was made under UV at 254 nm.

For determination of the sensitivities of the PCR assays, two-folddilutions of purified genomic DNA were used to determine the minimalnumber of genome copies which can be detected.

Results

Amplifications with the Staphylococcus genus-specific PCR assay. Thespecificity of the assay was assessed by performing 30-cycle and40-cycle PCR amplifications with the panel of gram-positive (47 speciesfrom 8 genera) and gram-negative (33 species from 22 genera) bacterialspecies listed in Table 12. The PCR assay was able to detect efficiently27 of 27 staphylococcal species tested in both 30-cycle and 40-cycleregimens. For 30-cycle PCR, all bacterial species tested other thanstaphylococci were negative. For 40-cycle PCR, Enterococcus faecalis andMacrococcus caseolyticus were slightly positive for theStaphylococcus-specific PCR assay. The other species tested remainednegative. Ubiquity tests performed on a collection of 295 clinicalisolates provided by the microbiology laboratory of the CentreHospitalier Universitaire de Québec, Pavillon Centre Hospitalier del'Université Laval (CHUL), including Staphylococcus aureus (n=34), S.auricularis (n=2), S. capitis (n=19), S. cohnii (n=5), S. epidermidis(n=18), S. haemolyticus (n=21), S. hominis (n=73), S. lugdunensis(n=17), S. saprophyticus (n=6), S. simulans (n=3), S. warneri (n=32) andStaphylococcus sp. (n=65), showed a uniform amplification signal withthe 30-cycle PCR assays and a perfect relation between the genotype andclassical identification schemes.

The sensitivity of the Staphylococcus-specific assay with 30-cycle and40-cycle PCR protocols was determined by using purified genomic DNA fromthe 11 staphylococcal species previously mentioned. For PCR with 30cycles, a detection limit of 50 copies of genomic DNA was consistentlyobtained. In order to enhance the sensitivity of the assay, the numberof cycles was increased. For 40-cycle PCR assays, the detection limitwas lowered to a range of 5-10 genome copies, depending on thestaphylococcal species tested.

Hybridization between the Staphylococcus-specific 371-bp amplicon andspecies-specific or genus-specific internal probes. Inter-speciespolymorphism was sufficient to generate species-specific internal probesfor each of the principal species involved in human diseases (S. aureus,S. epidermidis, S. haemolyticus, S. hominis and S. saprophyticus). Inorder to verify the intra-species sequence conservation of thenucleotide sequence, sequence comparisons were performed on the 371-bpamplicon from five unrelated ATCC and clinical strains for each of the 5principal staphylococcal species: S. aureus, S. epidermidis, S.haemolyticus, S. hominis and S. saprophyticus. Results showed a highlevel of conservation of nucleotide sequence between different unrelatedstrains from the same species. This sequence information allowed thedevelopment of staphylococcal species identification assays usingspecies-specific internal probes hybridizing to the 371-bp amplicon.These assays are specific and ubiquitous for those five staphylococcalspecies. In addition to the species-specific internal probes, thegenus-specific internals probes were able to recognize all or mostStaphylococcus species tested.

Example 8 Differentiating Between the Two Closely Related Yeast SpeciesCandida albicans and Candida Dubliniensis

It is often useful for the clinician to be able to differentiate betweentwo very closely related species of microorganisms. Candida albicans isthe most important cause of invasive human mycose. In recent years, avery closely related species, Candida dubliniensis, was isolated inimmunosuppressed patients. These two species are difficult todistinguish by classic biochemical methods. This example demonstratesthe use of tuf sequences to differentiate Candida albicans and Candidadubliniensis. PCR primers SEQ ID NOs. 11-12, from previous patentpublication WO98/20157, were selected for their ability to specificallyamplify a tuf (elongation factor 1 alpha type) fragment from bothspecies (see Annex XI for primer positions). Within this tuf fragment, aregion differentiating C. albicans and C. dubliniensis by twonucleotides was selected and used to design two internal probes (seeTable 49 for probe design, SEQ ID NOs. 577 and 578) specific for eachspecies. Amplification of genomic DNA from C. albicans and C.dubliniensis was carried out using DIG-11-dUTP as described above in thesection on chemiluminescent detection of amplification products.Internal probes SEQ ID NOs. 577 and 578 were immobilized on the bottomof individual microtiter plates and hybridization was carried out asdescribed above in the above section on chemiluminescent detection ofamplification products. Luminometer data showed that the amplicon fromC. albicans hybridized only to probe SEQ ID NO. 577 while the ampliconfrom C. dubliniensis hybridized only to probe SEQ ID NO. 578, therebydemonstrating that each probe was species-specific.

Example 9 Specific Identification of Entamoeba histolytica

Upon analysis of tuf (elongation factor 1 alpha) sequence data, it waspossible to find four regions where Entamoeba histolytica sequencesremained conserved while other parasitical and eukaryotic species havediverged. Primers TEntG38 (SEQ ID NO. 703), TEntG442 (SEQ ID NO. 704),TEntG534 (SEQ ID NO. 705), and TEntG768 (SEQ ID NO. 706) were designedso that SEQ ID NO. 703 could be paired with the three other primers. OnPTC-200 thermocyclers (MJ Research), the cycling conditions for initialsensitivity and specificity testing were 3 min. at 94° C. for initialdenaturation, then forty cycles of two steps consisting of 1 second at95° C. and 30 seconds at 55° C., followed by terminal extension at 72°C. for 2 minutes. Detection of the PCR products was made byelectrophoresis in agarose gels (2%) containing 0.25 μg/ml of ethidiumbromide. The three primer pairs could detect the equivalent of less than200 E. histolytica genome copies. Specificity was tested using 0.5 ng ofpurified genomic DNA from a panel of microorganisms including BabesiaBovis, Babesia microtti, Candida albicans, Crithidia fasciculata,Leishmania major, Leishmania hertigi and Neospora caninum. Only E.histolytica DNA could be amplified, thereby suggesting that the assaywas species-specific.

Example 10 Sensitive Identification of Chlamydia trachomatis

Upon analysis of tuf sequence data, it was possible to find two regionswhere Chlamydia trachomatis sequences remained conserved while otherspecies have diverged. Primers Ctr82 (SEQ ID NO. 554) and Ctr249 (SEQ IDNO. 555) were designed. With the PTC-200 thermocyclers (MJ Research),the optimal cycling conditions for maximum sensitivity and specificitywere determined to be 3 min. at 94° C. for initial denaturation, thenforty cycles of two steps consisting of 1 second at 95° C. and 30seconds at 60° C., followed by terminal extension at 72° C. for 2minutes. Detection of the PCR products was made by electrophoresis inagarose gels (2%) containing 0.25 μg/ml of ethidium bromide. The assaycould detect the equivalent of 8 C. trachomatis genome copies.Specificity was tested with 0.1 ng of purified genomic DNA from a panelof microorganisms including 22 species commonly encountered in thevaginal flora (Bacillus subtilis, Bacteroides fragilis, Candidaalbicans, Clostridium difficile, Corynebacterium cervicis,Corynebacterium urealyticum, Enterococcus faecalis, Enterococcusfaecium, Escherichia coli, Fusobacterium nucleatum, Gardnerellavaginalis, Haemophilus influenzae, Klebsiella oxytoca, Lactobacillusacidophilus, Peptococcus niger, Peptostreptococcus prevotii,Porphyromonas asaccharolytica, Prevotella melaminogenica,Propionibacterium acnes, Staphylococcus aureus, Streptococcusacidominimus, and Streptococcus agalactiae). Only C. trachomatis DNAcould be amplified, thereby suggesting that the assay wasspecies-specific.

Example 11 Genus-Specific Detection and Identification of Enterococci

Upon analysis of tuf sequence data and comparison with the repertory oftuf sequences, it was possible to find two regions where Enterococcussequences remained conserved while other genera have diverged (Table65). Primer pair Encg313dF and Encg599c (SEQ ID NOs. 1137 and 1136) wastested for its specificity by using purified genomic DNA from a panel ofbacteria listed in Table 10. Using the PTC-200 thermocycler (MJResearch), the optimal cycling conditions for maximum sensitivity andspecificity were determined to be 3 min. at 94° C. for initialdenaturation, then forty cycles of two steps consisting of 1 second at95° C. and 30 seconds at 55° C., followed by terminal extension at 72°C. for 2 minutes. Detection of the PCR products was made byelectrophoresis in agarose gels (2%) containing 0.25 μg/ml of ethidiumbromide. Visualization of the PCR products was made under UV at 254 nm.The 18 enterococcal species listed in Table 10 were all amplifiedefficiently. The only other species amplified were Abiotrophia adiacens,Gemella haemolysans and Gemella morbillorum, three gram-positivespecies. Sensitivity tested with several strains of E. casseliflavus, E.faecium, E. faecalis, E. flavescens and E. gallinarum and with onestrain of each other Enterococcus species listed in Table 10 ranged from1 to 10 copies of genomic DNA. The sequence variation within the 308-bpamplicon was sufficient so that internal probes could be used tospeciate the amplicon and differenciate enterococci from Abiotrophiaadiacens, Gemella haemolysans and Gemella morbillorum, thereby allowingto achieve excellent specificity. Species-specific internal probes weregenerated for each of the clinically important species, E. faecalis (SEQID NO. 1174), E. faecium (SEQ ID NO. 602), and the group including E.casseliflavus, E. flavescens and E. gallinarum (SEQ ID NO. 1122) (Table66). The species-specific internal probes were able to differentiatetheir respective Enterococcus species from all other Enterococcusspecies. These assays are sensitive, specific and ubiquitous for thosefive Enterococcus species.

Example 12 Identification of the Major Bacterial Platelets ContaminantsUsing Tuf Sequences with a Multiplex PCR Test

Blood platelets preparations need to be monitored for bacterialcontaminations. The tuf sequences of 17 important bacterial contaminantsof platelets were aligned. As shown in Table 57, analysis of thesesequences allowed the design of PCR primers. Since in the case ofcontamination of platelet concentrates, detecting all species (not justthe more frequently encountered ones) is desirable, perfect specificityof primers was not an issue in the design. However, sensitivity isimportant. That is why, to avoid having to put too much degeneracy, onlythe most frequent contaminants were included in primer design, knowingthat the selected primers would anyway be able to amplify more speciesthan the 17 used in the design because they target highly conservedregions of tuf sequences. Oligonucleotide sequences which are conservedin these 17 major bacterial contaminants of platelet concentrates werechosen (oligos Tplaq 769 and Tplaq 991, respectively SEQ ID NOs. 636 and637) thereby permitting the detection of these bacterial species.However, sensitivity was slightly deficient with staphylococci. Toensure maximal sensitivity in the detection of all the more frequentbacterial contaminants, a multiplex assay also including oligonucleotideprimers targetting the Staphylococcus genera (oligos Stag 422, SEQ IDNO. 553; and Stag 765, SEQ ID NO. 575) was developed. The bacterialspecies detected with the assay are listed in Table 14.

The primer pairs, oligos SEQ ID NO. 636 and SEQ ID NO. 637 that give anamplification product of 245 pb, and oligos SEQ ID NO. 553 and SEQ IDNO. 575 that give an amplification product of 368 pb, were usedsimultaneously in the multiplex PCR assay. Detection of these PCRproducts was made on the LightCycler thermocycler (Idaho Technology)using SYBR® Green I (Molecular Probe Inc.). SYBR® Green I is afluorescent dye that binds specifically to double-stranded DNA.

Fluorogenic detection of PCR products with the LightCycler was carriedout using 1.0 μM of both Tplaq primers (SEQ ID NOs. 636-637) and 0.4 μMof both TStaG primers (SEQ ID NOs. 553 and 575), 2.5 mM MgCl₂, BSA 7.5μM, dNTP 0.2 mM (Pharmacia), 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 0.5 UTaq DNA polymerase (Boerhinger Mannheim) coupled with TaqStart™ antibody(Clontech), and 0.07 ng of genomic DNA sample in a final volume of 7 Theoptimal cycling conditions for maximum sensitivity and specificity were1 minute at 94° C. for initial denaturation, then forty-five cycles ofthree steps consisting of 0 second at 95° C., 5 seconds at 60° C. and 9seconds at 72° C. Amplification was monitored during each elongationcycle by measuring the level of SYBR® Green I. However, real analysistakes place after PCR. Melting curves are done for each sample andtransformation of the melting peak allows determination of Tm. Thusprimer-dimer and specific PCR product are discriminated. With thisassay, all prominent bacterial contaminants of platelet concentrateslisted in Table 57 and Table 14 were detected. Sensitivity tests wereperformed on the 9 most frequent bacterial contaminants of platelets.The detection limit was less than 20 genome copies for E. cloacae, B.cereus, S. choleraesuis and S. marcescens; less than 15 genome copiesfor P. aeruginosa; and 2 to 3 copies were detected for S. aureus, S.epidermidis, E. coli and K. pneumoniae. Further refinements of assayconditions should increase sensitivity levels.

Example 13 The Resolving Power of the tuf and atpD Sequences Databasesis Comparable to the Biochemical Methods for Bacterial Identification

The present gold standard for bacterial identification is mainly basedon key morphological traits and batteries of biochemical tests. Here wedemonstrate that the use of tuf and atpD sequences combined with simplephylogenetic analysis of databases formed by these sequences iscomparable to the gold standard. In the process of acquiring data forthe tuf sequences, we sequenced the tuf gene of a strain that was givento us labelled as Staphylococcus hominis ATCC 35982. That tuf sequence(SEQ ID NO. 192) was incorporated into the tuf sequences database andsubjected to a basic phylogenic analysis using the Pileup command fromversion 10 of the GCG package (Genetics Computer Group). This analysisindicated that SEQ ID NO. 192 is not associated with other S. hominisstrains but rather with the S. warneri strains. The ATCC 35982 strainwas sent to the reference laboratory of the Laboratoire de santépublique du Québec (LSPQ). They used the classic identification schemefor staphylococci (Kloos and Schleifer, 1975., J. Clin. Microbiol.1:82-88). Their results shown that although the colonial morphologycould correspond to S. hominis, the more precise biochemical assays didnot. These assays included discriminant mannitol, mannose and riboseacidification tests as well as rapid and dense growth in deepthioglycolate agar. The LSPQ report identified strain ATCC 35982 as S.warneri which confirms our database analysis. The same thing happenedfor S. warneri (SEQ ID NO. 187) which had initially been identified asS. haemolyticus by a routine clinical laboratory using a low resolvingpower automated system (MicroScan, AutoScan-4™). Again, the tuf and LSPQanalysis agreed on its identification as S. warneri. In numerous otherinstances, in the course of acquiring tuf and atpD sequence data fromvarious species and genera, analysis of our tuf and/or atpD sequencedatabases permitted the exact identification of mislabelled orerroneously identified strains. These results clearly demonstrate theusefulness and the high resolving power of our sequence-basedidentification assays using the tuf and atpD sequences databases.

Example 14 Detection Of Group B Streptococci From Clinical Specimens*

Introduction

Streptococcus agalactiae, the group B streptococcus (GBS), isresponsible for a severe illness affecting neonate infants. Thebacterium is passed from the healthy carrier mother to the baby duringdelivery. To prevent this infection, it is recommended to treatexpectant mothers susceptible of carrying GBS in their vaginal/analflora. Carrier status is often a transient condition and rigorousmonitoring requires cultures and classic bacterial identification weeksbefore delivery. To improve the detection and identification of GBS wedeveloped a rapid, specific and sensitive PCR test fast enough to beperformed right at delivery.

Materials and Methods

GBS clinical specimens. A total of 66 duplicate vaginal/anal swabs werecollected from 41 consenting pregnant women admitted for delivery at theCentre Hospitalier Universitaire de Québec, Pavillon Saint-Francoisd'Assise following the CDC recommendations. The samples were obtainedeither before or after rupture of membranes. The swab samples weretested at the Centre de Recherche en Infectiologie de l'Université Lavalwithin 24 hours of collection. Upon receipt, one swab was cut and thenthe tip of the swab was added to GNS selective broth for identificationof group B streptococci (GBS) by the standard culture methodsrecommended by the CDC. The other swab was processed following theinstruction of the IDI DNA extraction kit (Infectio Diagnotics (IDI)Inc.) prior to PCR amplification.

Oligonucleotides. PCR primers, Tsag340 (SEQ ID NO. 549) and Tsag552 (SEQID NO. 550) complementary to the regions of the tuf gene unique for GBSwere designed based upon a multiple sequence alignment using ourrepertory of tuf sequences. Oligo primer analysis software (version 5.0)(National Biosciences) was used to analyse primers annealingtemperature, secondary structure potential as well as mispriming anddimerization potential. The primers were synthesized using a model 391DNA synthesizer (Applied Biosystems).

A pair of fluorescently labeled adjacent hybridization probes Sag465-F(SEQ ID NO. 583) and Sag436-C (SEQ ID NO. 582) were synthesized andpurified by Operon Technologies. They were designed to meet therecommendations of the manufacturer (Idaho Technology) and based uponmultiple sequence alignment analysis using our repertory of tufsequences to be specific and ubiquitous for GBS. These adjacent probes,which are separated by one nucleotide, allow fluorescence resonanceenergy transfer (FRET), generating an increased fluorescence signal whenboth hybridized simultaneously to their target sequences. The probe SEQID NO. 583 was labeled with FITC in 3 prime while SEQ ID NO. 582 waslabeled with Cy5 in 5 prime. The Cy5-labeled probes contained a3′-blocking phosphate group to prevent extension of the probes duringthe PCR reactions.

PCR amplification. Conventional amplifications were performed eitherfrom 2 μl of a purified genomic DNA preparation or cell lysates ofvaginal/anal specimens. The 20 μl PCR mixture contained 0.4 μM of eachGBS-specific primer (SEQ ID NOs. 549-550), 200 μM of eachdeoxyribonucleotide (Pharmacia Biotech), 10 mM Tris-HCl (pH 9.0), 50 mMKCl, 0.1% Triton X-100, 2.5 mM MgCl₂, 3.3 mg/ml bovine serum albumin(BSA) (Sigma), and 0.5 U of Taq polymerase (Promega) combined with theTaqStart™ antibody (Clontech). The TaqStart™ antibody, which is aneutralizing monoclonal antibody of Taq DNA polymerase, was added to allPCR reactions to enhance the efficiency of the amplification. The PCRmixtures were subjected to thermal cycling (3 min at 95° C. and then 40cycles of 1 s at 95° C., and 30 s at 62° C. with a 2-min final extensionat 72° C.) with a PTC-200 DNA Engine thermocycler (MJ research). ThePCR-amplified reaction mixture was resolved by agarose gelelectrophoresis.

The LightCycler™ PCR amplifications were performed with 1 μl of apurified genomic DNA preparation or cell lysates of vaginal/analspecimens. The 100 amplification mixture consisted of 0.4 μM eachGBS-specific primer (SEQ ID NOs. 549-550), 200 μM each dNTP, 0.2 μM eachfluorescently labeled probe (SEQ ID NOs. 582-583), 300 μg/ml BSA(Sigma), and 1 μl of 10×PC2 buffer (containing 50 mM Tris-HCl (pH 9.1),16 mM ammonium sulfate, 3.5 mM Mg²⁺, and 150 μg/ml BSA) and 0.5 UKlenTaq1™ (AB Peptides) coupled with TaqStart™ antibody (Clontech).KlenTaq1™ is a highly active and more heat-stable DNA polymerase without5′-exonuclease activity. This prevents hydrolysis of hybridized probesby the 5′ to 3′ exonuclease activity. A volume of 7 μl of the PCRmixture was transferred into a composite capillary tube (IdahoTechnology). The tubes were then centrifuged to move the reactionmixture to the tips of the capillaries and then cleaned withoptical-grade methanol. Subsequently the capillaries were loaded intothe carousel of a LC32 LightCycler™ (Idaho Technology), an instrumentthat combines rapid-cycle PCR with fluorescence analysis for continuousmonitoring during amplification. The PCR reaction mixtures weresubjected to a denaturation step at 94° C. for 3 min followed by 45cycles of 0 s at 94° C., 20 s at 64° C. and 10 s at 72° C. with atemperature transition rate of 20° C./s. Fluorescence signals wereobtained at each cycle by sequentially positioning each capillary on thecarousel at the focus of optical elements affiliated to the built-influorimeter for 100 milliseconds. Complete amplification and analysisrequired about 35 min.

Specificity and sensitivity tests. The specificity of the conventionaland LightCycler™ PCR assays was verified by using purified genomic DNA(0.1 ng/reaction) from a battery of ATCC reference strains representing35 clinically relevant gram-positive species (Abiotrophia defectiva ATCC49176, Bifidobacterium breve ATCC 15700, Clostridium difficile ATCC9689, Corynebacterium urealyticum ATCC 43042, Enterococcus casseliflavusATCC 25788, Enterococcus durans ATCC 19432, Enterococcus faecalis ATCC29212, Enterococcus faecium ATCC 19434, Enterococcus gallinarum ATCC49573, Enterococcus raffinosus ATCC 49427, Lactobacillus reuteri ATCC23273, Lactococcus lactis ATCC 19435, Listeria monocytogenes ATCC 15313,Peptococcus niger ATCC 27731, Peptostreptococcus anaerobius ATCC 27337,Peptostreptococcus prevotii ATCC 9321, Staphylococcus aureus ATCC 25923,Staphylococcus epidermidis ATCC 14990, Staphylococcus haemolyticus ATCC29970, Staphylococcus saprophyticus ATCC 15305, Streptococcus agalactiaeATCC 27591, Streptococcus anginosus ATCC 33397, Streptococcus Bovis ATCC33317, Streptococcus constellatus ATCC 27823, Streptococcus dysgalactiaeATCC 43078, Streptococcus gordonii ATCC 10558, Streptococcus mitis ATCC33399, Streptococcus mutans ATCC 25175, Streptococcus oralis ATCC 35037,Streptococcus parauberis ATCC 6631, Streptococcus pneumoniae ATCC 6303,Streptococcus pyogenes ATCC 19615, Streptococcus salivarius ATCC 7073,Streptococcus sanguinis ATCC 10556, Streptococcus uberis ATCC 19436).These microbial species included 15 species of streptococci and manymembers of the normal vaginal and anal floras. In addition, 40 GBSisolates of human origin, whose identification was confirmed by a latexagglutination test (Streptex, Murex), were also used to evaluate theubiquity of the assay.

For determination of the sensitivities (i.e., the minimal number ofgenome copies that could be detected) for conventional and LightCycler™PCR assays, serial 10-fold or 2-fold dilutions of purified genomic DNAfrom 5 GBS ATCC strains were used.

Results

Evaluation of the GBS-specific conventional and LightCycler™ PCR assays.The specificity of the two assays demonstrated that only DNAs from GBSstrains could be amplified. Both PCR assays did not amplify DNAs fromany other bacterial species tested including 14 streptococcal speciesother than GBS as well as phylogenetically related species belonging tothe genera Enterococcus, Peptostreptococcus and Lactococcus. Importantmembers of the vaginal or anal flora, including coagulase-negativestaphylococci, Lactobacillus sp., and Bacteriodes sp. were also negativewith the GBS-specific PCR assay. The LightCycler™ PCR assays detectedonly GBS DNA by producing an increased fluorescence signal which wasinterpreted as a positive PCR result. Both PCR methods were able toamplify all of 40 GBS clinical isolates, showing a perfect correlationwith the phenotypic identification methods.

The sensitivity of the assay was determined by using purified genomicDNA from the 5 ATCC strains of GBS. The detection limit for all of these5 strains was one genome copy of GBS. The detection limit of the assaywith the LightCycler™ was 3.5 fg of genomic DNA (corresponding to 1-2genome copies of GBS). These results confirmed the high sensitivity ofour GBS-specific PCR assay.

Direct Detection of GBS from vaginal/anal specimens. Among 66vaginal/anal specimens tested, 11 were positive for GBS by both cultureand PCR. There was one sample positive by culture only. The sensitivityof both PCR methods with vaginal/anal specimens for identifyingcolonization status in pregnant women at delivery was 91.7% whencompared to culture results. The specificity and positive predictivevalues were both 100% and the negative predictive value was 97.8%. Thetime for obtaining results was approximately 45 min for LightCycler™PCR, approximately 100 min for conventional PCR and 48 hours forculture.

Conclusion

We have developed two PCR assays (conventional and LightCycler™) for thedetection of GBS, which are specific (i.e., no amplification of DNA froma variety of bacterial species other than GBS) and sensitive (i.e., ableto detect around 1 genome copy for several reference ATCC strains ofGBS). Both PCR assays are able to detect GBS directly from vaginal/analspecimens in a very short turnaround time. Using the real-time PCR assayon LightCycler™, we can detect GBS carriage in pregnant women atdelivery within 45 minutes.

Example 15 Simultaneous Detection and Identification of Streptococcuspyogenes and its Pyrogenic Exotoxin A

The rapid detection of Streptococcus pyogenes and of its pyrogenicexotoxin A is of clinical importance. We developed a multiplex assaywhich permits the detection of strains of S. pyogenes carrying thepyrogenic toxin A gene, which is associated with scarlet fever and otherpathologies. In order to specifically detect S. pyogenes, nucleotidesequences of the pyrrolidone carboxylyl peptidase (pcp) gene werealigned to design PCR primers Spy291 (SEQ ID NO. 1211) and Spy473 (SEQID NO. 1210). Next, we designed primers for the specific detection ofthe pyrogenic exotoxin A. Nucleotide sequences of the speA gene, carriedon the bacteriophage T12, were aligned as shown in Table 60 to designPCR primers Spytx814 (SEQ ID NO. 994) and Spytx 927 (SEQ ID NO. 995).

The primer pairs: oligos SEQ ID NOs. 1210-1211, yielding anamplification product of 207 bp, and oligos SEQ ID NOs. 994-995,yielding an amplification product of 135 bp, were used in a multiplexPCR assay.

PCR amplification was carried out using 0.4 μM of both pairs of primers,2.5 mM MgCl₂, BSA 0.05 μM, dNTP 0.2 μM (Pharmacia), 10 mM Tris-HCl (pH9.0), 0.1% Triton X-100, 2.5 mM MgCl₂, 0.5 U Taq DNA polymerase(Promega) coupled with TaqStart™ antibody (Clontech Laboratories Inc.),and 1 μl of genomic DNA sample in a final volume of 20 μl. PCRamplification was performed using a PTC-200 thermal cycler (MJResearch). The optimal cycling conditions for maximum specificity andsensitivity were 3 minutes at 94° C. for initial denaturation, thenforty cycles of two steps consisting of 1 second at 95° C. and 30seconds at 63° C., followed by a final step of 2 minutes at 72° C.Detection of the PCR products was made by electrophoresis in agarosegels (2%) containing 0.25 μg/ml of ethidium bromide. Visualization ofthe PCR products was made under UV at 254 nm.

The detection limit was less than 5 genome copies for both S. pyogenesand its pyrogenic exotoxin A. The assay was specific for pyrogenicexotoxin A-producing S. pyogenes: strains of the 27 other species ofStreptococcus tested, as well as 20 strains of various gram-positive andgram-negative bacterial species were all negative.

A similar approach was used to design an alternative set ofspeA-specific primers (SEQ ID NOs. 996 to 998, see Table 62). Inaddition, another set of primers based on the tuf gene (SEQ ID NOs. 999to 1001, see Annex XXV) could be used to specifically detectStreptococcus pyogenes.

Example 16 Real-Time Detection and Identification of ShigaToxin-Producing Bacteria

Shiga toxin-producing Escherichia coli and Shigella dysenteriae causebloody diarrhea. Currently, identification relies mainly on thephenotypic identification of S. dysenteriae and E. coli serotypeO157:H7. However, other serotypes of E. coli are increasingly found tobe producers of type 1 and/or type 2 Shiga toxins. Two pairs of PCRprimers targeting highly conserved regions present in each of the Shigatoxin genes stx₁ and stx₂ were designed to amplify all variants of thosegenes (see Tables 64 and 65). The first primer pair, oligonucleotides1SLT224 (SEQ ID NO. 1081) and 1SLT385 (SEQ ID NO. 1080), yields anamplification product of 186 bp from the stx₁ gene. For this amplicon,the 1SLTB1-Fam (SEQ ID NO. 1084) molecular beacon was designed for thespecific detection of stx/using the fluorescent label6-carboxy-fluorescein. The 1SltS1-FAM (SEQ ID NO. 2012) molecularscorpion was also designed as an alternate way for the specificdetection of stx₁. A second pair of PCR primers, oligonucleotides2SLT537 (SEQ ID NO. 1078) and 2SLT678b (SEQ ID NO. 1079), yields anamplification product of 160 bp from the stx₂ gene. Molecular beacon2SLTB1-Tet (SEQ ID NO. 1085) was designed for the specific detection ofstx₂ using the fluorescent label 5-tetrachloro-fluorescein. Both primerpairs were combined in a multiplex PCR assay.

PCR amplification was carried out using 0.8 μM of primer pair SEQ IDNOs. 1080-1081, 0.5 μM of primer pair SEQ ID NOs. 1078-1079, 0.3 μM ofeach molecular beacon, 8 mM MgCl₂, 490 μg/mL BSA, 0.2 mM dNTPs(Pharmacia), 50 mM Tris-HCl, 16 mM NH₄SO₄, 1× TaqMaster (Eppendorf), 2.5U KlenTaq1 DNA polymerase (AB Peptides) coupled with TaqStart™ antibody(Clontech Laboratories Inc.), and 1 μl of genomic DNA sample in a finalvolume of 25 μl. PCR amplification was performed using a SmartCyclerthermal cycler (Cepheid). The optimal cycling conditions for maximumsensitivity and specificity were 60 seconds at 95° C. for initialdenaturation, then 45 cycles of three steps consisting of 10 seconds at95° C., 15 seconds at 56° C. and 5 seconds at 72° C. Detection of thePCR products was made in real-time by measuring the fluorescent signalemitted by the molecular beacon when it hybridizes to its target at theend of the annealing step at 56° C.

The detection limit was the equivalent of less than 5 genome copies. Theassay was specific for the detection of both toxins, as demonstrated bythe perfect correlation between PCR results and the phenotypiccharacterization performed using antibodies specific for each Shigatoxin type. The assay was successfully performed on several Shigatoxin-producing strains isolated from various geographic areas of theworld, including 10 O157:H7 E. coli, 5 non-O157:H7 E. coli and 4 S.dysenteriae.

Example 17 Development of a PCR Assay for the Detection andIdentification of Staphylococci at Genus and Species Levels and itsAssociated mecA Gene

The Staphylococcus-specific PCR primers described in Example 7 (SEQ IDNOs. 553 and 575) were used in multiplex with the mecA-specific PCRprimers and the S. aureus-specific primers described in our assignedU.S. Pat. No. 5,994,066 (SEQ ID NOs. 261 and 262 for mecA and SEQ IDNOs. 152 and 153 for S. aureus in the said patent). Sequence alignmentanalysis of 10 publicly available mecA gene sequences allowed to designan internal probe specific to mecA (SEQ ID NO. 1177). An internal probewas also designed for the S. aureus-specific amplicon (SEQ ID NO 1234).PCR amplification and agarose gel electrophoresis of the amplifiedproducts were performed as described in Example 7, with the exceptionthat 0.4 μM (each) of the two Staphylococcus-specific primers (SEQ IDNOs. 553 and 575) and 0.4 μM (each) of the mecA-specific primers and 0.4μM (each) of the S. aureus-specific primers were used in the PCRmixture. The specificity of the multiplex assay with 40-cycle PCRprotocols was verified by using purified genomic DNA from fivemethicillin-resistant and fifteen methicillin-sensitive staphylococcalstrains. The sensitivity of the multiplex assay with 40-cycle PCRprotocols was determined by using purified genomic DNA from twenty-threemethicillin-resistant and twenty-eight methicillin-sensitivestaphylococcal strains. The detection limit was 2 to 10 genome copies ofgenomic DNA, depending on the staphylococcal species tested.Furthermore, the mecA-specific internal probe, the S. aureus-specificinternal probe and the coagulase-negative staphylococci-specificinternal probe (described in Example 7) were able to recognizetwenty-three methicillin-resistant staphylococcal strains andtwenty-eight methicillin-sensitive staphylococcal strains with highsensitivity and specificity.

The format of the assay is not limited to the one described above. Aperson skilled in the art could adapt the assay for different formatssuch as PCR with real-time detection using molecular beacon probes.Molecular beacon probes designed to be used in this assay include, butare not limited to, SEQ ID NO. 1232 for detection of the S.aureus-specific amplicon, SEQ ID NO. 1233 for detection ofcoagulase-negative staphylococci and SEQ ID NO. 1231 for detection ofmecA.

Alternatively, a multiplex PCR assay containing theStaphylococcus-specific PCR primers described in Example 7 (SEQ ID NOs.553 and 575) and the mecA-specific PCR primers described in our assignedU.S. Pat. No. 5,994,066 (SEQ ID NOs. 261 and 262 in the said patent)were developed. PCR amplification and agarose gel electrophoresis of theamplified products were performed as described in Example 7, with theexception that 0.4 μM (each) of the Staphylococcus-specific primers (SEQID NOs. 553 and 575) and 0.4 μM (each) of the mecA-specific primersdescribed in our assigned U.S. Pat. No. 5,994,066 (SEQ ID NOs. 261 and262 in the said patent) were used in the PCR mixture. The sensitivity ofthe multiplex assay with 40-cycle PCR protocols was determined by usingpurified genomic DNA from two methicillin-resistant and fivemethicillin-sensitive staphylococcal strains. The detection limit was 2to 5 copies of genomic DNA, depending on the staphylococcal speciestested. The specificity of the multiplex PCR assay coupled withcapture-probe hybridization was tested with two strains ofmethicillin-resistant S. aureus, two strains of methicillin-sensitive S.aureus and seven strains of methicillin-sensitive coagulase-negativestaphylococci. The mecA-specific internal probe (SEQ ID NO. 1177) andthe S. aureus-specific internal probe (SEQ ID NO. 587) described inExample 7 were able to recognize all the strains with high specificityshowing a perfect correlation with susceptibility to methicillin. Thesensitivity of the PCR assay coupled with capture-probe hybridizationwas tested with one strain of methicillin-resistant S. aureus. Thedetection limit was around 10 copies of genomic DNA.

Example 18 Sequencing of pbp1a, pbp2b and pbp2x genes of Streptoccoccuspneumoniae

Penicillin resistance in Streptococcus pneumoniae involves thesequential alteration of up to five penicillin-binding proteins (PBPs)1A, 1B, 2A, 2× and 2B in such a way that their affinity is greatlyreduce toward the antibiotic molecule. The altered PBP genes have arisenas the result of interspecies recombination events from relatedstreptococcal species. Among the PBPs usually found in S. pneumoniae,PBPs 1A, 2B, and 2× play the most important role in the development ofpenicillin resistance. Alterations in PBP 2B and 2× mediate low-levelresistance to penicillin while additional alterations in PBP 1A plays asignificant role in full penicillin resistance.

In order to generate a database for pbp sequences that can be used fordesign of primers and/or probes for the specific and ubiquitousdetection of β-lactam resistance in S. pneumoniae, pbp1a, pbp2b andpbp2x DNA fragments sequenced by us or selected from public databases(GenBank and EMBL) from a variety of S. pneumoniae strains were used todesign oligonucleotide primers. This database is essential for thedesign of specific and ubiquitous primers and/or probes for detection ofβ-lactam resistance in S. pneumoniae since the altered PBP 1A, PBP 2Band PBP 2× of β-lactam resistant S. pneumoniae are encoded by mosaicgenes with numerous sequence variations among resistant isolates. ThePCR primers were located in conserved regions of pbp genes and were ableto amplify pbp1a, pbp2b, and pbp2× sequences of several strains of S.pneumoniae having various levels of resistance to penicillin andthird-generation cephalosporins. Using primer pairs SEQ ID NOs. 1125 and1126, SEQ ID NOs. 1142 and 1143, SEQ ID NOs. 1146 and 1147, it waspossible to amplify and determine pbp1a sequences SEQ ID NOs. 1004-1018,1648, 2056-2060 and 2062-2064, pbp2b sequences SEQ ID NOs. 1019-1033,and pbp2× sequences SEQ ID NOs. 1034-1048. Six other PCR primers (SEQ IDNOs. 1127-1128, 1144-1145, 1148-1149) were also designed and used tocomplete the sequencing of pbp1a, pbp2b and pbp2x amplificationproducts. The described primers (SEQ ID NOs. 1125 and 1126, SEQ ID NOs.1142 and 1143, SEQ ID NOs. 1146 and 1147, SEQ ID NOs. 1127-1128,1144-1145, 1148-1149) represent a powerful tool for generating new pbpsequences for design of primers and/or probes for detection of β-lactamresistance in S. pneumoniae.

Example 19 Sequencing of hexA Genes of Streptococcus Species

The hexA sequence of S. pneumoniae described in our assigned U.S. Pat.No. 5,994,066 (SEQ ID NO. 31 in the said patent, SEQ ID NO. 1183 in thepresent application) allowed the design of a PCR primer (SEQ ID NO.1182) which was used with primer Spn1401 described in our assigned U.S.Pat. No. 5,994,066 (SEQ ID NO. 156 in the said patent, SEQ ID NO. 1179in the present application) to generate a database for hexA sequencesthat can be used to design primers and/or probes for the specificidentification and detection of S. pneumoniae (Table 80). Using primersSEQ ID NO. 1179 and SEQ ID NO. 1182 (Annex XIII), it was possible toamplify and determine the hexA sequence from S. pneumoniae (4 strains)(SEQ ID NOs. 1184-1187), S. mitis (three strains) (SEQ ID NOs.1189-1191) and S. oralis (SEQ ID NO. 1188).

Example 20 Development of Multiplex PCR Assays Coupled with CaptureProbe Hybridization for the Detection and Identification ofStreptococcus pneumoniae and its Penicillin Resistance Genes.

Two different assays were developed to identify S. pneumoniae and itssusceptibility to penicillin.

Assay I:

Bacterial strains. The specificity of the multiplex PCR assay wasverified by using a panel of ATCC (American Type Culture Collection)reference strains consisting of 33 gram-negative and 67 gram-positivebacterial species (Table 13). In addition, a total of 98 strains of S.pneumoniae, 16 strains of S. mitis and 3 strains of S. oralis from theAmerican Type Culture Collection, the microbiology laboratory of theCentre Hospitalier Universitaire de Québec, Pavillon Centre Hospitalierde l'Université Laval (CHUL), (Step-Foy, Québec, Canada), theLaboratoire de santé publique du Québec, (Sainte-Anne-de-Bellevue,Québec, Canada), the Sunnybrook and Women's College Health SciencesCentre (Toronto, Canada), the Infectious Diseases Section, Department ofVeterans Affairs Medical Center, (Houston, USA) were also tested tofurther validate the Streptococcus pneumoniae-specific PCR assay. Thepenicillin MICs (minimal inhibitory concentrations) were measured by thebroth dilution method according to the recommended protocol of NCCLS.

PCR primers and internal probes. The analysis of hexA sequences from avariety of streptococcal species from the publicly available hexAsequence and from the database described in Example 19 (SEQ ID NOs.1184-1191) allowed the selection of a PCR primer specific to S.pneumoniae, SEQ ID NO. 1181. This primer was used with the S.pneumoniae-specific primer SEQ ID NO. 1179 to generate an amplificationproduct of 241 bp (Table 80). The PCR primer SEQ ID NO. 1181 is located127 nucleotides downstream on the hexA sequence compared to the originalS. pneumoniae-specific PCR primer Spn1515 described in our assigned U.S.Pat. No. 5,994,066 (SEQ ID NO. 157 in the said patent). Thesemodifications were done to ensure the design of the S.pneumoniae-specific internal probe according to the new hexA sequencesof several streptococcal species from the database described in Example19 (SEQ ID NOs. 1184-1191).

The analysis of pbp1a sequences from S. pneumoniae strains with variouslevels of penicillin resistance from public databases and from thedatabase described in Example 18 allowed the identification of aminoacid substitutions Ile-459 to Met and Ser-462 to Ala that occur inisolates with high-level penicillin resistance (MICs≧1 μg/ml), and aminoacid substitutions Ser-575 to Thr, Gln-576 to Gly and Phe-577 to Tyrthat are common to all penicillin-resistant isolates with MICs≧0.25μg/ml. As shown in Table 69, PCR primer pair SEQ ID NOs. 1130 and 1131were designed to detect high-level penicillin resistance (MICs≧1 μg/ml),whereas PCR primer pair SEQ ID NOs. 1129 and 1131 were designed todetect intermediate- and high-level penicillin resistance (MICs 0.25μg/ml).

The analysis of hexA sequences from the publicly available hexA sequenceand from the database described in Example 19 allowed the design of aninternal probe specific to S. pneumoniae (SEQ ID NO. 1180) (Table 80).The range of mismatches between the S. pneumoniae-specific 241-bpamplicon was from 2 to 5, in the middle of the 19-bp probe. The analysisof pbp1a sequences from public databases and from the database describedin Example 18 allowed the design of five internal probes containing allpossible mutations to detect the high-level penicillin resistance 383-bpamplicon (SEQ ID NOs. 1197, 1217-1220). Alternatively, two otherinternal probes (SEQ ID NOs. 2024-2025) can also be used to detect thehigh-level penicillin resistance 383-bp amplicon. Five internal probescontaining all possible mutations to detect the 157-bp amplicon whichincludes intermediate- and high-level penicillin resistance were alsodesigned (SEQ ID NOs. 1094, 1192-1193, 1214 and 1216). Design andsynthesis of primers and probes, and detection of the probehybridization were performed as described in Example 7. Table 69illustrates one of the internal probe for detection of the high-levelpenicillin resistance 383-bp amplicon (SEQ ID NO. 1197) and one of theinternal probe for detection of the intermediate- and high-levelpenicillin resistance 157-bp amplicon (SEQ ID NO. 1193).

PCR amplification. For all bacterial species, amplification wasperformed from purified genomic DNA using a PTC-200 thermocycler (MJResearch). 1 μl of genomic DNA at 0.1 ng/μl, or 1 μl of a bacteriallysate, was transferred to a 19 μl PCR mixture. Each PCR reactioncontained 50 mM KCl, 10 mM Tris-HCl (H 9.0), 0.1% Triton X-100, 2.5 mMMgCl₂, 0.1 μM (each) of the S. pneumoniae-specific primers SEQ ID NO.1179 and SEQ ID NO. 1181, 0.2 μM of primer SEQ ID NO. 1129, 0.7 μM ofprimer SEQ ID NO. 1131, and 0.6 μM of primer SEQ ID NO. 1130, 0.05 mMbovine serum albumin (BSA), and 0.5 U Taq polymerase (Promega) coupledwith TaqStart™ antibody. In order to generate Digoxigenin (DIG)-labeledamplicons for capture probe hybridization, 0.1×PCR DIG labeling fourdeoxynucleoside triphosphates mix (Boehringer Mannheim GmbH) was usedfor amplification.

For determination of the sensitivity of the PCR assays, 10-folddilutions of purified genomic DNA were used to determine the minimalnumber of genome copies which can be detected.

Capture probe hybridization. The DIG-labeled amplicons were hybridizedto the capture probes bound to 96-well plates. The plates were incubatedwith anti-DIG-alkaline phosphatase and the chemiluminescence wasmeasured by using a luminometer (MLX, Dynex Technologies Inc.) afterincubation with CSPD and recorded as Relative Light Unit (RLU). The RLUratio of tested sample with and without captures probes was thencalculated. A ratio≧2.0 was defined as a positive hybridization signal.All reactions were performed in duplicate.

Results

Amplifications with the multiplex PCR assay. The specificity of theassay was assessed by performing 40-cycle PCR amplifications with thepanel of gram-positive (67 species from 12 genera) and gram-negative (33species from 17 genera) bacterial species listed in Table 13. Allbacterial species tested other than S. pneumoniae were negative exceptS. mitis and S. oralis. Ubiquity tests were performed using a collectionof 98 S. pneumoniae strains including high-level penicillin resistance(n=53), intermediate resistance (n=12) and sensitive (n=33) strains.There was a perfect correlation between PCR and standard susceptibilitytesting for 33 penicillin-sensitive isolates. Among 12 S. pneumoniaeisolates with intermediate penicillin resistance based on susceptibilitytesting, 11 had intermediate resistance based on PCR, but one S.pneumoniae isolate with penicillin MIC of 0.25 μg/ml showed a high-levelpenicillin resistance based on genotyping. Among 53 isolates withhigh-level penicillin resistance based on susceptibility testing, 51 hadhigh-level penicillin resistance based on PCR but two isolates withpenicillin MIC >1 μg/ml showed an intermediate penicillin resistancebased on genotyping. In general, there was a good correlation betweenthe genotype and classical culture method for bacterial identificationand susceptibility testing.

The sensitivity of the S. pneumoniae-specific assay with 40-cycle PCRprotocols was determined by using purified genomic DNA from 9 isolatesof S. pneumoniae. The detection limit was around 10 copies of genomicDNA for all of them.

Post-PCR hybridization with internal probes. The specificity of themultiplex PCR assay coupled with capture-probe hybridization was testedwith 98 strains of S. pneumoniae, 16 strains of S. mitis and 3 strainsof S. oralis. The internal probe specific to S. pneumoniae (SEQ ID NO.1180) detected all 98 S. pneunoniae strains but did not hybridize to theS. mitis and S. oralis amplicons. The five internal probes specific tothe high-level resistance amplicon (SEQ ID NOs. 1197, 1217-1220)detected all amplification patterns corresponding to high-levelresistance. The two S. pneumoniae strains with penicillin MIC >1 μg/mlthat showed an intermediate penicillin resistance based on PCRamplification were also intermediate resistance based on probehybridization. Similarly, among 12 strains with intermediate-penicillinresistance based on susceptibility testing, 11 showedintermediate-penicillin resistance based on hybridization with the fiveinternal probes specific to the intermediate and high-level resistanceamplicon (SEQ ID NOs. 1094, 1192-1193, 1214 and 1216). The straindescribed above having a penicillin MIC of 0.25 μg/ml which washigh-level penicillin resistance based on PCR amplification was alsohigh-level resistance based on probe hybridization. In summary, thecombination of the multiplex PCR and hybridization assays results in ahighly specific test for the detection of penicillin-resistantStreptococcus pneumoniae.

Assay II:

Bacterial strains. The specificity of the multiplex PCR assay wasverified by using the same strains as those used for the development ofAssay I. The penicillin MICs (minimal inhibitory concentrations) weremeasured by the broth dilution method according to the recommendedprotocol of NCCLS.

PCR primers and internal probes. The analysis of pbp1a sequences from S.pneumoniae strains with various levels of penicillin resistance frompublic databases and from the database described in Example 18 allowedthe design of two primers located in the constant region of pbp1a. PCRprimer pair (SEQ ID NOs. 2015 and 2016) was designed to amplify a 888-bpvariable region of pbp1a from all S. pneumoniae strains. A series ofinternal probes were designed for identification of the pbp1a mutationsassociated with penicillin resistance in S. pneumoniae. For detection ofhigh-level penicillin resistance (MICs≧1 μg/ml), three internal probeswere designed (SEQ ID NOs. 2017-2019). Alternatively, ten other internalprobes were designed that can also be used for detection of high-levelresistance within the 888-bp pbp1a amplicon: (1) three internal probesfor identification of the amino acid substitutions Thr-371 to Ser or Alawithin the motif S370TMK (SEQ ID NOs. 2031-2033); (2) two internalprobes for detection of the amino acid substitutions Ile-459 to Met andSer-462 to Ala near the motif S428RN (SEQ ID NOs. 1135 and 2026); (3)two internal probes for identification of the amino acid substitutionsAsn-443 to Asp (SEQ ID NOs. 1134 and 2027); and (4) three internalprobes for detection of all sequence variations within another region(SEQ ID NOs. 2028-2030). For detection of high-level and intermediatepenicillin resistance (MICs 0.25 μg/ml), four internal probes weredesigned (SEQ ID NOs. 2020-2023). Alternatively, six other internalprobes were designed for detection of the four consecutive amino acidsubstitutions T574SQF to A574TGY near the motif K557TG (SEQ ID NOs.2034-2039) that can also be used for detection of intermediate- andhigh-level resistance within the 888-bp pbp1a amplicon.

PCR amplification. For all bacterial species, amplification wasperformed from purified genomic DNA using a PTC-200 thermocycler (MJResearch). 1 μl of genomic DNA at 0.1 ng/μl, or 1 μl of a bacteriallysate, was transferred to a 19 μl PCR mixture. Each PCR reactioncontained 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mMMgCl₂, 0.08 μM (each) of the S. pneumoniae-specific primers SEQ ID NO.1179 and SEQ ID NO. 1181, 0.4 μM of the pbp1a-specific primer SEQ ID NO.2015, 1.2 μM of pbp1a-specific primer SEQ ID NO. 2016, 0.05 mM bovineserum albumin (BSA), and 0.5 U Taq polymerase (Promega) coupled withTaqStart™ antibody. In order to generate Digoxigenin (DIG)-labeledamplicons for capture probe hybridization, 0.1×PCR DIG labeling fourdeoxynucleoside triphosphates mix (Boehringer Mannheim GmbH) was usedfor amplification.

For determination of the sensitivities of the PCR assays, 10-folddilutions of purified genomic DNA were used to determine the minimalnumber of genome copies which can be detected.

Capture probe hybridization. The DIG-labeled amplicons were hybridizedto the capture probes bound to 96-well plates as described for Assay I.

Results

Amplifications with the multiplex PCR assay. The specificity of theassay was assessed by performing 40-cycle PCR amplifications with thepanel of gram-positive (67 species from 12 genera) and gram-negative (33species from 17 genera) bacterial species listed in Table 13. Allbacterial species tested other than S. pneumoniae were negative exceptS. mitis and S. oralis. Ubiquity tests were performed using a collectionof 98 S. pneumoniae strains including high-level penicillin resistance(n=53), intermediate resistance (n=12) and sensitive (n=33) strains. Allthe above S. pneumoniae strains produced the 888-bp ampliconcorresponding to pbp1a and the 241-bp fragment corresponding to hexA.

The sensitivity of the S. pneumoniae-specific assay with 40-cycle PCRprotocols was determined by using purified genomic DNA from 9 isolatesof S. pneumoniae. The detection limit was around 10 copies of genomicDNA for all of them.

Post-PCR hybridization with internal probes. The specificity of themultiplex PCR assay coupled with capture-probe hybridization was testedwith 98 strains of S. pneumoniae, 16 strains of S. mitis and 3 strainsof S. oralis. The internal probe specific to S. pneumoniae (SEQ ID NO.1180) detected all 98 S. pneunoniae strains but did not hybridize to theS. mitis and S. oralis amplicons. The three internal probes (SEQ ID NOs2017-2019) specific to high-level resistance detected all the 43 strainswith high-level penicillin resistance based on susceptibility testing.Among 12 isolates with intermediate-penicillin resistance based onsusceptibility testing, 11 showed intermediate-penicillin resistancebased on hybridization with 4 internal probes (SEQ ID NOs. 2020-2023)and one strain having penicillin MIC of 0.25 μg/ml was misclassified ashigh-level penicillin resistance. In summary, the combination of themultiplex PCR and hybridization assays results in a highly specific testfor the detection of penicillin-resistant Streptococcus pneumoniae.

Example 21 Sequencing of the Vancomycin Resistance vanA, vanC1, vanC2and vanC3 Genes

The publicly available sequences of the vanH-vanA-vanX-vanY locus oftransposon Tn1546 from E. faecalis, vanC1 sequence from one strain of E.gallinarum, vanC2 and vanC3 sequences from a variety of E. casseliflavusand E. flavescens strains, respectively, allowed the design of PCRprimers able to amplify the vanA, vanC1, vanC2 and vanC3 sequences ofseveral Enterococcus species. Using primer pairs van6877 and van9106(SEQ ID NOs. 1150 and 1155), vanC1-122 and vanC1-1315 (SEQ ID NOs. 1110and 1109), and vanC2C3-1 and vanC2C3-1064 (SEQ ID NOs. 1108 and 1107),it was possible to amplify and determine vanA sequences SEQ ID NOs.1049-1057, vanC1 sequences SEQ ID NOs. 1058-1059, vanC2 sequences SEQ IDNOs. 1060-1063 and vanC3 sequences SEQ ID NOs. 1064-1066, respectively.Four other PCR primers (SEQ ID NOs. 1151-1154) were also designed andused to complete the sequencing of vanA amplification products.

Example 22 Development of a PCR Assay for the Detection andIdentification of Enterococci at Genus and Species Levels and itsAssociated Resistance Genes vanA and vanB

The comparison of vanA and vanB sequences revealed conserved regionsallowing the design of PCR primers specific to both vanA and vanBsequences (Annex Table 76). The PCR primer pair vanAB459 and vanAB830R(SEQ ID NOs. 1112 and 1111) was used in multiplex with theEnterococcus-specific primers Encg313dF and Encg599c (SEQ ID NOs. 1137and 1136) described in Example 11. Sequence alignment analysis of vanAand vanB sequences revealed regions suitable for the design of internalprobes specific to vanA (SEQ ID NO. 1170) and vanB (SEQ ID NO. 1171).PCR amplification and agarose gel electrophoresis of the amplifiedproducts were performed as described in Example 11. The optimal cyclingconditions for maximum sensitivity and specificity were found to be 3min. at 94° C., followed by forty cycles of two steps consisting of 1second at 95° C. and 30 seconds at 62° C., plus a terminal extension at72° C. for 2 minutes. The specificity of the multiplex assay with40-cycle PCR was verified by using 0.1 nanogram of purified genomic DNAfrom a panel of bacteria listed in Table 10. The sensitivity of themultiplex assay with 40-cycle PCR was verified with three strains of E.casseliflavus, eight strains of E. gallinarum, two strains of E.flavescens, two vancomycin-resistant strains of E. faecalis and onevancomycin-sensitive strain of E. faecalis, three vancomycin-resistantstrains of E. faecium, one vancomycin-sensitive strain of E. faecium andone strain of each of the other enterococcal species listed in Table 10.The detection limit was 1 to 10 copies of genomic DNA, depending on theenterococcal species tested. The vanA- and vanB-specific internal probes(SEQ ID NOs. 1170 and 1171), as well as the E. faecalis-and E.faecium-specific internal probes (SEQ ID NOs. 1174 and 602) and theinternal probe specific to the group including E. casseliflavus, E.gallinarum and E. flavescens (SEQ ID NO. 1122) described in Example 11,were able to recognize vancomycin-resistant enterococcal species withhigh sensitivity, specificity and ubiquity showing a perfect correlationbetween the genotypic and phenotypic analysis.

The format of the assay is not limited to the one described above. Aperson skilled in the art could adapt the assay for different formatssuch as PCR with real-time detection using molecular beacon probes.Molecular beacon probes designed to be used in this assay include, butare not limited to, SEQ ID NO. 1236 for the detection of E. faecalis,SEQ ID NO. 1235 for the detection of E. faecium, SEQ ID NO. 1240 for thedetection of vanA, and SEQ ID NO. 1241 for the detection of vanB.

Example 23 Development of a Multiplex PCR Assay for Detection andIdentification of Vancomycin-Resistant Enterococcus faecalis,Enterococcus faecium and the Group including Enterococcus Gallinarum,Enterococcus Casseliflavus, and Enterococcus flavescens

The analysis of vanA and vanB sequences revealed conserved regionsallowing design of a PCR primer pair (SEQ ID NOs. 1089 and 1090)specific to vanA sequences (Table 66) and a PCR primer pair (SEQ ID NOs.1095 and 1096) specific to vanB sequences (Table 67). The vanA-specificPCR primer pair (SEQ ID NOs. 1089 and 1090) was used in multiplex withthe vanB-specific PCR primer pair described in our assigned U.S. Pat.No. 5,994,066 (SEQ ID NOs. 1095 and 1096 in the present patent and SEQID NOs. 231 and 232 in the said patent). The comparison of vanC1, vanC2and vanC3 sequences revealed conserved regions allowing design of PCRprimers (SEQ ID NOs. 1101 and 1102) able to generate a 158-bp ampliconspecific to the group including E. gallinarum, E. casseliflavus and E.flavescens (Table 68). The vanC-specific PCR primer pair (SEQ ID NOs.1101 and 1102) was used in multiplex with the E. faecalis-specific PCRprimer pair described in our assigned U.S. Pat. No. 5,994,066 (SEQ IDNOs. 40 and 41 in the said patent) and with the E. faecium-specific PCRprimer pair described in our patent publication WO_(—)98/20157 (SEQ IDNOs. 1 and 2 in the said publication). For both multiplexes, the optimalcycling conditions for maximum sensitivity and specificity were found tobe 3 min. at 94° C., followed by forty cycles of two steps consisting of1 second at 95° C. and 30 seconds at 58° C., plus a terminal extensionat 72° C. for 2 minutes. Detection of the PCR products was made byelectrophoresis in agarose gels (2%) containing 0.25 μg/ml of ethidiumbromide. The vanA-specific PCR primer pair (SEQ ID NOs. 1089 and 1090),the vanB-specific primer pair (SEQ ID NOs. 1095 and 1096) and thevanC-specific primer pair (SEQ ID NOs. 1101 and 1102) were tested fortheir specificity by using 0.1 nanogram of purified genomic DNA from apanel of 5 vancomycin-sensitive Enterococcus species, 3vancomycin-resistant Enterococcus species, 13 other gram-positivebacteria and one gram-negative bacterium. Specificity tests wereperformed with the E. faecium-specific PCR primer pair described in ourpatent publication WO 98/20157 (SEQ ID NOs. 1 and 2 in the saidpublication) and with the E. faecalis-specific PCR primer pair describedin our assigned U.S. Pat. No. 5,994,066 (SEQ ID NOs. 40 and 41 in thesaid patent) on a panel of 37 gram-positive bacterial species. AllEnterococcus strains were amplified with high specificity showing aperfect correlation between the genotypic and phenotypic analysis. Thesensitivity of the assays was determined for several strains of E.gallinarum, E. casseliflavus, E. flavescens and vancomycin-resistant E.faecalis and K. faecium. Using each of the E. faecalis-and E.faecium-specific PCR primer pairs as well as vanA-, vanB- andvanC-specific PCR primers used alone or in multiplex as described above,the sensitivity ranged from 1 to 10 copies of genomic DNA.

The format of the assay is not limited to the one described above. Aperson skilled in the art could adapt the assay for different formatssuch as PCR with real-time detection using molecular beacon probes.Molecular beacon probes designed to be used in this assay include, butare not limited to, SEQ ID NO. 1238 for the detection of E. faecalis,SEQ ID NO. 1237 for the detection of E. faecium, SEQ ID NO. 1239 for thedetection of vanA, and SEQ ID NO. 1241 for the detection of vanB.

Alternatively, another PCR assay was developed for the detection ofvancomycin-resistant E. faecium and vancomycin-resistant E. faecalis.This assay included two multiplex: (1) the first multiplex contained thevanA-specific primer pair (SEQ ID NOs. 1090-1091) and the vanB-specificPCR primer pair described in our assigned U.S. Pat. No. 5,994,066 (SEQID NOs. 1095 and 1096 in the present patent and SEQ ID NOs. 231 and 232in the said patent), and (2) the second multiplex contained the E.faecalis-specific PCR primer pair described in our assigned U.S. Pat.No. 5,994,066 (SEQ ID NOs. 40 and 41 in the said patent) and the E.faecium-specific PCR primer pair described in our patent publicationWO98/20157 (SEQ ID NOs. 1 and 2 in the said publication). For bothmultiplexes, the optimal cycling conditions for maximum sensitivity andspecificity were found to be 3 min. at 94° C., followed by forty cyclesof two steps consisting of 1 second at 95° C. and 30 seconds at 58° C.,plus a terminal extension at 72° C. for 2 minutes. Detection of the PCRproducts was made by electrophoresis in agarose gels (2%) containing0.25 μg/ml of ethidium bromide. The two multiplexes were tested fortheir specificity by using 0.1 nanogram of purified genomic DNA from apanel of two vancomycin-sensitive E. faecalis strains, twovancomycin-resistant E. faecalis strains, two vancomycin-sensitive E.faecium strains, two vancomycin-resistant E. faecium strains, 16 otherenterococcal species and 31 other gram-positive bacterial species. Allthe E. faecium and E. faecalis strains were amplified with highspecificity showing a perfect correlation between the genotypic analysisand the susceptibility to glycopeptide antibiotics (vancomycin andteicoplanin). The sensitivity of the assay was determined for twovancomycin-resistant E. faecalis strains and two vancomycin-resistant E.faecium strains. The detection limit was 5 copies of genomic DNA for allthe strains.

This multiplex PCR assay was coupled with capture-probe hybridization.Four internal probes were designed: one specific to the vanA amplicon(SEQ ID NO. 2292), one specific to the vanB amplicon (SEQ ID NO. 2294),one specific to the E. faecalis amplicon (SEQ ID NO. 2291) and onespecific to the E. faecium amplicon (SEQ ID NO. 2287). Each of theinternal probes detected their specific amplicons with high specificityand sensitivity.

Example 24 Universal Amplification Involving the EF-G (fusA) Subdivisionof tuf Sequences

As shown in FIG. 3, primers SEQ ID NOs. 1228 and 1229 were designed toamplify the region between the end of fusA and the beginning of tufgenes in the str operon. Genomic DNAs from a panel of 35 strains weretested for PCR amplification with those primers. In the initialexperiment, the following strains showed a positive result: Abiotrophiaadiacens ATCC 49175, Abiotrophia defectiva ATCC 49176, Bacillus subtilisATCC 27370, Closridium difficile ATCC 9689, Enterococcus avium ATCC14025, Enterococcus casseliflavus ATCC 25788, Enterococcus cecorum ATCC43198, Enterococcus faecalis ATCC 29212, Enterococcus faecium ATCC19434, Enterococcus flavescens ATCC 49996, Enterococcus gallinarum ATCC49573, Enterococcus solitarius ATCC 49428, Escherichia coli ATCC 11775,Haemophilus influenzae ATCC 9006, Lactobacillus acidophilus ATCC 4356,Peptococcus niger ATCC 27731, Proteus mirabilis ATCC 25933,Staphylococcus aureus ATCC 43300, Staphylococcus auricularis ATCC 33753,Staphylococcus capitis ATCC 27840, Staphylococcus epidemidis ATCC 14990,Staphylococcus haemolyticus ATCC 29970, Staphylococcus hominis ATCC27844, Staphylococcus lugdunensis ATCC 43809, Staphylococcussaprophyticus ATCC 15305, Staphylococcus simulans ATCC 27848, andStaphylococcus warneri ATCC 27836. This primer pair could amplifyadditional bacterial species; however, there was no amplification forsome species, suggesting that the PCR cycling conditions could beoptimized or the primers modified. For example, SEQ ID NO. 1227 wasdesigned to amplify a broader range of species.

In addition to other possible primer combinations to amplify the regioncovering fusA and tuf, FIG. 3 illustrates the positions of amplificationprimers SEQ ID NOs. 1221-1227 which could be used for universalamplification of fusA segments. All of the above mentioned primers (SEQID NOs. 1221-1229) could be useful for the universal and/or the specificdetection of bacteria.

Moreover, different combinations of primers SEQ ID NOs. 1221-1229,sometimes in combination with tuf sequencing primer SEQ ID NO. 697, wereused to sequence portions of the str operon, including the intergenicregion. In this manner, the following sequences were generated: SEQ IDNOs. 1518-1526, 1578-1580, 1786-1821, 1822-1834, 1838-1843, 2184, 2187,2188, 2214-2249, and 2255-2269.

Example 25 DNA Fragment Isolation from Staphylococcus saprophyticus byArbitrarily Primed PCR

DNA sequences of unknown coding potential for the species-specificdetection and identification of Staphylococcus saprophyticus wereobtained by the method of arbitrarily primed PCR (AP-PCR).

AP-PCR is a method which can be used to generate specific DNA probes formicroorganisms (Fani et al., 1993, Molecular Ecology 2:243-250). Adescription of the AP-PCR protocol used to isolate a species-specificgenomic DNA fragment from Staphylococcus saprophyticus follows. Twentydifferent oligonucleotide primers of 10 nucleotides in length (allincluded in the AP-PCR kit OPAD (Operon Technologies, Inc., Alameda,Calif.)) were tested systematically with DNAs from 5 bacterial strainsof Staphylococcus saprophyticus as well as with bacterial strains of 27other staphylococcal (non-S. saprophyticus) species. For all bacterialspecies, amplification was performed directly from one μL (0.1 ng/μL) ofpurified genomic DNA. The 25 μL PCR reaction mixture contained 50 mMKCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM MgCl₂, 1.2 μM ofonly one of the 20 different AP-PCR primers OPAD, 200 μM of each of thefour dNTPs, 0.5 U of Taq DNA polymerase (Promega Corp., Madison, Wis.)coupled with TaqStart™ antibody (Clontech Laboratories Inc., Palo Alto,Calif.). PCR reactions were subjected to cycling using a MJ ResearchPTC-200 thermal cycler as follows: 3 min at 96° C. followed by 42 cyclesof 1 min at 94° C. for the denaturation step, 1 min at 31° C. for theannealing step and 2 min at 72° C. for the extension step. A finalextension step of 7 min at 72° C. followed the 42 cycles to ensurecomplete extension of PCR products. Subsequently, twenty microliters ofthe PCR-amplified mixture were resolved by electrophoresis on a 1.5%agarose gel containing 0.25 μg/ml of ethidium bromide. The size of theamplification products was estimated by comparison with a 50-bpmolecular weight ladder.

Amplification patterns specific for Staphylococcus saprophyticus wereobserved with the AP-PCR primer OPAD-16 (sequence: 5′-AACGGGCGTC-3′).Amplification with this primer consistently showed a band correspondingto a DNA fragment of approximately 380 bp for all Staphylococcussaprophyticus strains tested but not for any of the other staphylococcalspecies tested.

The band corresponding to the 380 bp amplicon, specific and ubiquitousfor S. saprophyticus based on AP-PCR, was excised from the agarose geland purified using the QIAquick™ gel extraction kit (QIAGEN Inc.). Thegel-purified DNA fragment was cloned into the T/A cloning site of thepCR 2.1™plasmid vector (Invitrogen Inc.) using T4 DNA ligase (NewEngland BioLabs). Recombinant plasmids were transformed into E. coliDH5a competent cells using standard procedures. All reactions wereperformed according to the manufacturer's instructions. Plasmid DNAisolation was done by the method of Birnboim and Doly (Nucleic AcidRes., 1979, 7:1513-1523) for small-scale preparations. All plasmid DNApreparations were digested with the EcoRI restriction endonuclease toensure the presence of the approximately 380 bp AP-PCR insert into theplasmid. Subsequently, a large-scale and highly purified plasmid DNApreparation was performed from two selected clones shown to carry theAP-PCR insert by using the QIAGEN plasmid purification kit (midiformat). These large-scale plasmid preparations were used for automatedDNA sequencing.

The 380 bp nucleotide sequence was determined for three strains of S.saprophyticus (SEQ ID NOs. 74, 1093, and 1198). Both strands of theAP-PCR insert from the two selected clones were sequenced by thedideoxynucleotide chain termination sequencing method with SP6 and T7sequencing primers by using the Applied Biosystems automated DNAsequencer (model 373A) with their PRISM™ Sequenase® TerminatorDouble-stranded DNA Sequencing Kit (Applied Biosystems, Foster City,Calif.).

Optimal species-specific amplification primers (SEQ ID NOs. 1208 and1209) have been selected from the sequenced AP-PCR Staphylococcussaprophyticus DNA fragments with the help of the primer analysissoftware Oligo™ 5.0 (National BioSciences Inc.). The selected primerswere tested in PCR assays to verify their specificity and ubiquity. Dataobtained with DNA preparations from reference ATCC strains of 49gram-positive and 31 gram-negative bacterial species, including 28different staphylococcal species, indicate that the selected primerpairs are specific for Staphylococcus saprophyticus since noamplification signal has been observed with DNAs from the otherstaphylococcal or bacterial species tested. This assay was able toamplify efficiently DNA from all 60 strains of S. saprophyticus fromvarious origins tested. The sensitivity level achieved for three S.saprophyticus reference ATCC strains was around 6 genome copies.

Example 26 Sequencing of Prokaryotic tuf Gene Fragments

The comparison of publicly available tuf sequences from a variety ofbacterial species revealed conserved regions, allowing the design of PCRprimers able to amplify tuf sequences from a wide range of bacterialspecies. Using primer pair SEQ ID NOs. 664 and 697, it was possible toamplify and determine tuf sequences SEQ ID NOs.: 1-73, 75-241, 607-618,621, 662, 675, 717-736, 868-888, 932, 967-989, 992, 1002, 1572-1575,1662-1663, 1715-1733, 1835-1837, 1877-1878, 1880-1881, 2183, 2185, 2200,2201, and 2270-2272.

Example 27 Sequencing of Procaryotic recA Gene Fragments

The comparison of publicly available recA sequences from a variety ofbacterial species revealed conserved regions, allowing the design of PCRprimers able to amplify recA sequences from a wide range of bacterialspecies. Using primer pairs SEQ ID NOs. 921-922 and 1605-1606, it waspossible to amplify and determine recA sequences SEQ ID NOs.: 990-991,1003, 1288-1289, 1714, 1756-1763, 1866-1873 and 2202-2212.

Example 28

Specific Detection and Identification of Escherichia coli/Shigella Sp.Using tuf Sequences

The analysis of tuf sequences from a variety of bacterial speciesallowed the selection of PCR primers (SEQ ID NOs. 1661 and 1665) and ofan internal probe (SEQ ID NO. 2168) specific to Escherichiacoli/Shigella sp. The strategy used to design the PCR primers was basedon the analysis of a multiple sequence alignment of various tufsequences. The multiple sequence alignment included the tuf sequences ofEscherichia coli/Shigella sp. as well as tuf sequences from otherspecies and bacterial genera, especially representatives of closelyrelated species. A careful analysis of this alignment allowed theselection of oligonucleotide sequences which are conserved within thetarget species but which discriminate sequences from other species,especially from the closely related species, thereby permitting thespecies-specific and ubiquitous detection and identification of thetarget bacterial species.

The chosen primer pair, oligos SEQ ID NOs. 1661 and 1665, gives anamplification product of 219 bp. Standard PCR was carried out using 0.4μM of each primer, 2.5 mM MgCl₂, BSA 0.05 mM, 50 mM KCl, 10 mM Tris-HCl(pH 9.0), 0.1% Triton X-100, dNTPs 0.2 mM (Pharmacia), 0.5 U Taq DNApolymerase (Promega) coupled with TaqStart™ antibody (ClontechLaboratories Inc.), 1 μl of genomic DNA sample in a final volume of 20μl using a PTC-200 thermocycler (MJ Research). The optimal cyclingconditions for maximum sensitivity and specificity were 3 minutes at 95°C. for initial denaturation, then forty cycles of two steps consistingof 1 second at 95° C. and 30 seconds at 60° C., followed by terminalextension at 72° C. for 2 minutes. Detection of the PCR products wasmade by electrophoresis in agarose gels (2%) containing 0.25 μg/ml ofethidium bromide. Visualization of the PCR products was made under UV at254 nm.

Specificity of the assay was tested by adding to the PCR reactions 0.1ng of genomic DNA from each of the following bacterial species:Escherichia coli (7 strains), Shigella sonnei, Shigella flexneri,Shigella dysenteriae, Salmonella typhimyurium, Salmonella typhi,Salmonella enteritidis, Tatumella ptyseos, Klebsiella pneumoniae (2strains), Enterobacter aerogenes, Citrobacter farmeri, Campylobacterjejuni, Serratia marcescens. Amplification was observed only for theEscherichia coli and Shigella sp. strains listed and Escherichiafergusonii. The sensitivity of the assay with 40-cycle PCR was verifiedwith one strain of E. coli and three strains of Shigella sp. Thedetection limit for E. coli and Shigella sp. was 1 to 10 copies ofgenomic DNA, depending on the strains tested.

Example 29 Specific Detection and Identification of Klebsiellapneumoniae Using atpD Sequences

The analysis of atpD sequences from a variety of bacterial speciesallowed the selection of PCR primers specific to K. pneumoniae. Theprimer design strategy is similar to the strategy described in Example28 except that atpD sequences were used in the alignment.

Two K. pneumoniae-specific primers were selected, (SEQ ID NOs. 1331 and1332) which give an amplification product of 115 bp. Standard PCR wascarried out on PTC-200 thermocyclers (MJ Research) using 0.4 μM of eachprimer as described in Example 28. The optimal cycling conditions formaximum sensitivity and specificity were as follow: three minutes at 95°C. for initial denaturation, then forty cycles of two steps consistingof 1 second at 95° C. and 30 seconds at 55° C., followed by terminalextension at 72° C. for 2 minutes.

Specificity of the assay was tested by adding to the PCR reactions 0.1ng of genomic DNA from each of the following bacterial species:Klebsiella pneumoniae (2 strains), Klebsiella ornitholytica, Klebsiellaoxytoca (2 strains), Klebsiella planticola, Klebsiella terrigena,Citrobacter freundii, Escherichia coli, Salmonella cholerasuis typhi,Serratia marcescens, Enterobacter aerogenes, Proteus vulgaris, Kluyveraascorbata, Kluyvera georgiana, Kluyvera cryocrescens and Yersiniaenterolitica. Amplification was detected for the two K. pneumoniaestrains, K. planticola, K. terrigena and the three Kluyvera speciestested. Analysis of the multiple alignment sequence of the atpD geneallowed the design of an internal probe SEQ ID NO. 2167 which candiscrimate Klebsiella pneumoniae from other Klebsiella sp. and Kluyverasp. The sensitivity of the assay with 40-cycle PCR was verified with onestrain of K. pneumoniae. The detection limit for K. pneumoniae wasaround 10 copies of genomic DNA.

Example 30 Specific Detection and Identification of AcinetobacterBaumannii Using atpD Sequences

The analysis of atpD sequences from a variety of bacterial speciesallowed the selection of PCR primers specific to Acinetobacterbaumannii. The primer design strategy is similar to the strategydescribed in Example 28.

Two A. baumannii-specific primers were selected, SEQ ID NOs. 1690 and1691, which give an amplification product of 233 bp. Standard PCR wascarried out on PTC-200 thermocyclers (MJ Research) using 0.4 μM of eachprimer as described in Example 28. The optimal cycling conditions formaximum sensitivity and specificity were as follow: three minutes at 95°C. for initial denaturation, then forty cycles of two steps consistingof 1 second at 95° C. and 30 seconds at 60° C., followed by terminalextension at 72° C. for 2 minutes.

Specificity of the assay was tested by adding to the PCR reactions 0.1ng of genomic DNA from each of the following bacterial species:Acinetobacter baumannii (3 strains), Acinetobacter anitratus,Acinetobacter lwoffi, Serratia marcescens, Enterobacter cloacae,Enterococcus faecalis, Pseudomonas aeruginosa, Psychrobacterphenylpyruvicus, Neisseria gonorrheoae, Haemophilus haemoliticus,Yersinia enterolitica, Proteus vulgaris, Eikenella corrodens,Escherichia coli. Amplification was detected only for A. baumannii, A.anitratus and A. lwoffi. The sensitivity of the assay with 40-cycle PCRwas verified with two strains of A. baumannii. The detection limit forthe two A. baumannii strains tested was 5 copies of genomic DNA.Analysis of the multiple alignment sequence of the atpD gene allowed thedesign of a A. baumannii-specific internal probe (SEQ ID NO. 2169).

Example 31 Specific Detection and Identification of Neisseriagonorrhoeae Using tuf Sequences

The analysis of tuf sequences from a variety of bacterial speciesallowed the selection of PCR primers specific to Neisseria gonorrhoeae.The primer design strategy is similar to the strategy described inExample 28.

Two N. gonorrhoeae-specific primers were selected, SEQ ID NOs. 551 and552, which give an amplification product of 139 bp. PCR amplificationwas carried out on PTC-200 thermocyclers (MJ Research) using 0.4 μM ofeach primer as described in Example 28. The optimal cycling conditionsfor maximum sensitivity and specificity were as follow: three minutes at95° C. for initial denaturation, then forty cycles of two stepsconsisting of 1 second at 95° C. and 30 seconds at 65° C., followed byterminal extension at 72° C. for 2 minutes.

Specificity of the assay was tested by adding into the PCR reactions,0.1 ng of genomic DNA from each of the following bacterial species:Neisseria gonorrhoeae (19 strains), Neisseria meningitidis (2 strains),Neisseria lactamica, Neisseria flavescens, Neisseria animalis, Neisseriacanis, Neisseria cuniculi, Neisseria elongata, Neisseria mucosa,Neisseria polysaccharea, Neisseria sicca, Neisseria subflava, Neisseriaweaveri. Amplification was detected only for N. gonorrhoeae, N. siccaand N. polysaccharea. The sensitivity of the assay with 40-cycle PCR wasverified with two strains of N. gonorrhoeae. The detection limit for theN. gonorrhoeae strains tested was 5 copies of genomic DNA. Analysis ofthe multiple alignment sequence of the tuf gene allowed the design of aninternal probe, SEQ ID NO. 2166, which can discriminate N. gonorrhoeaefrom N. sicca and N. polysaccharea.

Example 32 Sequencing of Bacterial gyrA and parC Gene Fragments.Sequencing of Bacterial gyrA and parC Fragments

One of the major mechanism of resistance to quinolone in variousbacterial species is mediated by target changes (DNA gyrase and/ortopoisomerase IV). These enzymes control DNA topology and are vital forchromosome function and replication. Each of these enzymes is a tetramercomposed of two subunits: GyrA and GyrB forming A₂B₂ complex in DNAgyrase; and ParC and ParE forming and C₂E₂ complex in DNA topoisomeraseIV. It has been shown that they are hotspots, called thequinolone-resistance-determining region (QRDR) for mutations within gyrAthat encodes for the GyrA subunit of DNA gyrase and within parC thatencodes the parC subunit of topoisomerase IV.

In order to generate a database for gyrA and parC sequences that can beused for design of primers and/or probes for the specific detection ofquinolone resistance in various bacterial species, gyrA and parC DNAfragments selected from public database (GenBanK and EMBL) from avariety of bacterial species were used to design oligonucleotideprimers.

Using primer pair SEQ ID NOs. 1297 and 1298, it was possible to amplifyand determine gyrA sequences from Klebsiella oxytoca (SEQ ID NO. 1764),Klebsiella pneumoniae subsp. ozaneae (SEQ ID NO. 1765), Klebsiellaplanticola (SEQ ID NO. 1766), Klebsiella pneumoniae (SEQ ID NO. 1767),Klebsiella pneumoniae subsp. pneumoniae (two strains) (SEQ ID NOs.1768-1769), Klebsiella pneumoniae subsp. rhinoscleromatis (SEQ ID NO.1770), Klebsiella terrigena (SEQ ID NO. 1771), Kluyvera ascorbata (SEQID NO. 2013), Kluyvera georgiana (SEQ ID NO. 2014) and Escherichia coli(4 strains) (SEQ ID NOs. 2277-2280). Using primer pair SEQ ID NOs. 1291and 1292, it was possible to amplify and determine gyrA sequences fromLegionella pneumophila subsp. pneumophila (SEQ ID NO. 1772), Proteusmirabilis (SEQ ID NO. 1773), Providencia rettgeri (SEQ ID NO. 1774),Proteus vulgaris (SEQ ID NO. 1775) and Yersinia enterolitica (SEQ ID NO.1776). Using primer pair SEQ ID NOs. 1340 and 1341, it was possible toamplify and determine gyrA sequence from Staphylococcus aureus (SEQ IDNO. 1255).

Using primers SEQ ID NOs. 1318 and 1319, it was possible to amplify anddetermine parC sequences from K. oxytoca (two strains) (SEQ ID NOs.1777-1778), Klebsiella pneumoniae subsp. ozaenae (SEQ ID NO. 1779),Klebsiella planticola (SEQ ID NO. 1780), Klebsiella pneumoniae (SEQ IDNO. 1781), Klebsiella pneumoniae subsp. pneumoniae (two strains) (SEQ IDNOs. 1782-1783), Klebsiella pneumoniae subsp. rhinoscleromatis (SEQ IDNO. 1784) and Klebsiella terrigena (SEQ ID NO. 1785).

Example 33 Development of a PCR Assay for the Specific Detection andIdentification of Staphylococcus aureus and its Quinolone ResistanceGenes GyrA and ParC

The analysis of gyrA and parC sequences from a variety of bacterialspecies revealed conserved regions allowing the design of PCR primersspecific to the quinolone-resistance-determining region (QRDR) of gyrAand parC from Staphylococcus aureus. PCR primer pair SEQ ID NOs. 1340and 1341 was designed to amplify the gyrA sequence of S. aureus, whereasPCR primer pair SEQ ID NOs. 1342 and 1343 was designed to amplify S.aureus parC. The comparison of gyrA and parC sequences from S. aureusstrains with various levels of quinolone resistance allowed theidentification of amino acid substitutions Ser-84 to Leu, Glu-88 to Glyor Lys in the GyrA subunit of DNA gyrase encoded by gyrA and amino acidchanges Ser-80 to Phe or Tyr and Ala-116 to Glu in the ParC subunit oftopoisomerase IV encoded by parC. These amino acid substitutions in GyrAand ParC subunits occur in isolates with intermediate- or high-levelquinolone resistance. Internal probes for the specific detection ofwild-type S. aureus gyrA (SEQ ID NO. 1940) and wild-type S. aureus parC(SEQ ID NO. 1941) as well as internal probes for the specific detectionof each of the gyrA (SEQ ID NOs. 1333-1335) and parC mutationsidentified in quinolone-resistant S. aureus (SEQ ID NOs. 1336-1339) weredesigned.

The gyrA- and parC-specific primer pairs (SEQ ID NOs. 1340-1341 and SEQID NOs. 1342-1343) were used in multiplex. PCR amplification was carriedout on PTC-200 thermocyclers (MJ Research) using 0.3, 0.3, 0.6 and 0.6μM of each primers, respectively, as described in Example 28. Theoptimal cycling conditions for maximum sensitivity and specificity were3 minutes at 95° C. for initial denaturation, then forty cycles of twosteps consisting of 1 second at 95° C. and 30 seconds at 62° C.,followed by terminal extension at 72° C. for 2 minutes. Detection of thePCR products was made by electrophoresis in agarose gels (2%) containing0.25 μg/ml of ethidium bromide. The specificity of the multiplex assaywith 40-cycle PCR was verified by using 0.1 ng of purified genomic DNAfrom a panel of gram-positive bacteria. The list included the following:Abiotrophia adiacens, Abiotrophia defectiva, Bacillus cereus, Bacillusmycoides, Enterococcus faecalis (2 strains), Enterococcus flavescens,Gemella morbillorum, Lactococcus lactis, Listeria innocua, Listeriamonocytogenes, Staphylococcus aureus (5 strains), Staphylococcusauricalis, Staphylococcus capitis subsp. urealyticus, Staphylococcuscarnosus, Staphylococcus chromogenes, Staphylococcus epidermidis (3strains), Staphylococcus gallinarum, Staphylococcus haemolyticus (2strains), Staphylococcus hominis, Staphylococcus hominis subsp hominis,Staphylococcuslentus, Staphylococcus lugdunensis, Staphylococcussaccharolyticus, Staphylococcus saprophyticus (3 strains),Staphylococcus simulans, Staphylococcus warneri, Staphylococcus xylosus,Streptococcus agalactiae, Streptococcus pneumoniae. Strong amplificationof both gyrA and parC genes was only detected for the S. aureus strainstested. The sensitivity of the multiplex assay with 40-cycle PCR wasverified with one quinolone-sensitive and four quinolone-resistantstrains of S. aureus. The detection limit was 2 to 10 copies of genomicDNA, depending on the strains tested.

Detection of the hybridization with the internal probes was performed asdescribed in Example 7. The internal probes specific to wild-type gyrAand parC of S. aureus and to the gyrA and parC variants of S. aureuswere able to recognize two quinolone-resistant and onequinolone-sensitive S. aureus strains showing a perfect correlation withthe susceptibility to quinolones.

The complete assay for the specific detection of S. aureus and itssusceptibility to quinolone contains the Staphylococcus-specific primers(SEQ ID NOs. 553 and 575) described in Example 7 and the multiplexcontaining the S. aureus gyrA- and parC-specific primer pairs (SEQ IDNOs. 1340-1341 and SEQ ID NOs. 1342-1343). Amplification is coupled withpost-PCR hybridization with the internal probe specific to S. aureus(SEQ ID NO. 587) described in Example 7 and the internal probes specificto wild-type S. aureus gyrA and parC (SEQ ID NOs. 1940-1941) and to theS. aureus gyrA and parC variants (SEQ ID NOs. 1333-1338).

An assay was also developed for the detection of quinolone-resistant S.aureus using the SmartCycler (Cepheid). Real-time detection is based onthe use of S. aureus parC-specific primers (SEQ ID NOs. 1342 and 1343)and the Staphylococcus-specific primers (SEQ ID NOs. 553 and 575)described in Example 7. Internal probes were designed for molecularbeacon detection of the wild-type S. aureus parC (SEQ ID NO.1939), fordetection of the Ser-80 to Tyr or Phe amino acid substitutions in theParC subunit encoded by S. aureus parC (SEQ ID NOs. 1938 and 1955) andfor detection of S. aureus (SEQ ID NO. 2282).

Example 34 Development of a PCR Assay for the Detection andIdentification of Klebsiella pneumoniae and Its Quinolone ResistanceGenes gyrA and parC

The analysis of gyrA and parC sequences from a variety of bacterialspecies from the public databases and from the database described inExample 32 revealed conserved regions allowing the design of PCR primersspecific to the quinolone-resistance-determining region (QRDR) of gyrAand parC from K. pneumoniae. PCR primer pair SEQ ID NOs. 1936 and 1937,or pair SEQ ID NOs. 1937 and 1942, were designed to amplify the gyrAsequence of K. pneumoniae, whereas PCR primer pair SEQ ID NOs. 1934 and1935 was designed to amplify K. pneumoniae parC sequence. An alternativepair, SEQ ID NOs. 1935 and 1936, can also amplify K. pneumoniae parC.The comparison of gyrA and parC sequences from K. pneumoniae strainswith various levels of quinolone resistance allowed the identificationof amino acid substitutions Ser-83 to Tyr or Phe and Asp-87 to Gly orAla and Asp-87 to Asn in the GyrA subunit of DNA gyrase encoded by gyrAand amino acid changes Ser-80 to Ile or Arg and Glu-84 to Gly or Lys inthe ParC subunit of topoisomerase IV encoded by parC. These amino acidsubstitutions in the GyrA and ParC subunits occur in isolates withintermediate- or high-level quinolone resistance. Internal probes forthe specific detection of wild-type K. pneumoniae gyrA (SEQ ID NO. 1943)and wild-type K. pneumoniae parC (SEQ ID NO. 1944) as well as internalprobes for the specific detection of each of the gyrA (SEQ ID NOs.1945-1949) and parC mutations identified in quinolone-resistant K.pneumoniae (SEQ ID NOs. 1950-1953) were designed.

Two multiplex using the K. pneumoniae gyrA- and parC-specific primerpairs were used: the first multiplex contained K. pneumoniaegyrA-specific primers (SEQ ID NOs. 1937 and 1942) and K. pneumoniaeparC-specific primers (SEQ ID NOs. 1934 and 1935) and the secondmultiplex contained K. pneumoniae gyrA/parC-specific primer (SEQ ID NOs.1936), K. pneumoniae gyrA-specific primer (SEQ ID NO. 1937) and K.pneumoniae parC-specific primer (SEQ ID NO. 1935). Standard PCR wascarried out on PTC-200 thermocyclers (MJ Research) using for the firstmultiplex 0.6, 0.6, 0.4, 0.4 μM of each primer, respectively, and forthe second multiplex 0.8, 0.4, 0.4 μM of each primer, respectively. PCRamplification and agarose gel electrophoresis of the amplified productswere performed as described in Example 28. The optimal cyclingconditions for maximum sensitivity and specificity were 3 minutes at 95°C. for initial denaturation, then forty cycles of two steps consistingof 1 second at 95° C. and 30 seconds at 62° C., followed by terminalextension at 72° C. for 2 minutes. The specificity of the two multiplexassays with 40-cycle PCR was verified by using 0.1 ng of purifiedgenomic DNA from a panel of gram-negative bacteria. The list included:Acinetobacter baumannii, Citrobacter freundii, Eikenella corrodens,Enterobacter aerogenes, Enterobacter cancerogenes, Enterobacter cloacae,Escherichia coli (10 strains), Haemophilus influenzae, Klebsiellapneumoniae, Klebsiella ornitholytica, Klebsiella oxytoca (2 strains),Klebsiella planticola, Klebsiella terrigena, Kluyvera ascorbata,Kluyvera cryocrescens, Kluyvera georgiana, Neisseria gonorrhoeae,Proteus mirabilis, Proteus vulgaris, Pseudomonas aeruginosa, Salmonellacholeraesuis subsp. typhimurium, Salmonella enteritidis, Serratialiquefaciens, Serratia marcescens and Yersinia enterocolytica. For bothmultiplex, strong amplification of both gyrA and parC was observed onlyfor the K. pneumoniae strain tested. The sensitivity of the twomultiplex assays with 40-cycle PCR was verified with onequinolone-sensitive strain of K. pneumoniae. The detection limit wasaround 10 copies of genomic DNA.

The complete assay for the specific detection of K. pneumoniae and itssusceptibility to quinolone contains the Klebsiella-specific primers(SEQ ID NOs. 1331 and 1332) described in Example 29 and either themultiplex containing the K. pneumoniae gyrA- and parC-specific primers(SEQ ID NOs. 1935, 1936, 1937) or the multiplex containing the K.pneumoniae gyrA- and parC-specific primers (SEQ ID NOs. 1934, 1937,1939, 1942). Amplification is coupled with post-PCR hybridization withthe internal probe specific to K. pneumoniae (SEQ ID NO. 2167) describedin Example 29 and the internal probes specific to wild-type K.pneumoniae gyrA and parC (SEQ ID NOs. 1943, 1944) and to the K.pneumoniae gyrA and parC variants (SEQ ID NOs. 1945-1949 and 1950-1953).

An assay was also developed for the detection of quinolone-resistant K.pneumoniae using the SmartCycler (Cepheid). Real-time detection is basedon the use of resistant K. pneumoniae gyrA-specific primers (SEQ ID NOs.1936 and 1937) and the K. pneumoniae-specific primers (SEQ ID NOs. 1331and 1332) described in Example 29. Internal probes were designed formolecular beacon detection of the wild-type K. pneumoniae gyrA (SEQ IDNO. 2251), for detection of the Ser-83 to Tyr or Phe and/or Asp-87 toGly or Asn in the GyrA subunit of DNA gyrase encoded by gyrA (SEQ IDNOs. 2250) and for detection of K. pneumoniae (SEQ ID NO. 2281).

Example 35 Development of a PCR Assay for Detection and Identificationof S. Pneumoniae and its Quinolone Resistance Genes gyrA and parC

The analysis of gyrA and parC sequences from a variety of bacterialspecies revealed conserved regions allowing the design of PCR primersable to amplify the quinolone-resistance-determining region (QRDR) ofgyrA and parC from all S. pneumoniae strains. PCR primer pair SEQ IDNOs. 2040 and 2041 was designed to amplify the QRDR of S. pneumoniaegyrA, whereas PCR primer pair SEQ ID NOs. 2044 and 2045 was designed toamplify the QRDR of S. pneumoniae parC. The comparison of gyrA and parCsequences from S. pneumoniae strains with various levels of quinoloneresistance allowed the identification of amino acid substitutions Ser-81to Phe or Tyr in the GyrA subunit of DNA gyrase encoded by gyrA andamino acid changes Ser-79 to Phe in the ParC subunit of topoisomerase IVencoded by parC. These amino acid substitutions in the GyrA and ParCsubunits occur in isolates with intermediate- or high-level quinoloneresistance. Internal probes for the specific detection of each of thegyrA (SEQ ID NOs. 2042 and 2043) and parC (SEQ ID NO. 2046) mutationsidentified in quinolone-resistant S. pneumoniae were designed.

For all bacterial species, amplification was performed from purifiedgenomic DNA. 1 μl of genomic DNA at 0.1 ng/1 μL, was transferreddirectly to a 19 μl PCR mixture. Each PCR reaction contained 50 mM KCl,10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM MgCl₂, 0.4 μM (each)of the above primers SEQ ID NOs. 2040, 2041, 2044 and 2045, 0.05 mMbovine serum albumin (BSA) and 0.5 U Taq polymerase coupled withTaqStart™ antibody. The optimal cycling conditions for maximumsensitivity and specificity were 3 minutes at 95° C. for initialdenaturation, then forty cycles of two steps consisting of 1 second at95° C. and 30 seconds at 58° C., followed by terminal extension at 72°C. for 2 minutes. In order to generate Digoxigenin (DIG)-labeledamplicons for capture probe hybridization, 0.1×PCR DIG labeling fourdeoxynucleoside triphosphates mix (Boehringer Mannheim GmbH) was usedfor amplification.

The DIG-labeled amplicons were hybridized to the capture probes bound to96-well plates. The plates were incubated with anti-DIG-alkalinephosphatase and the chemiluminescence was measured by using aluminometer (MLX, Dynex Technologies Inc.) after incubation with CSPDand recorded as Relative Light Unit (RLU). The RLU ratio of testedsample with and without captures probes was then calculated. A ratio 2.0was defined as a positive hybridization signal. All reactions wereperformed in duplicate.

The specificity of the multiplex assay with 40-cycle PCR was verified byusing 0.1 ng of purified genomic DNA from a panel of bacteria listed inTable 13. Strong amplification of both gyrA and parC was detected onlyfor the S. pneumoniae strains tested. Weak amplification of both gyrAand parC genes was detected for Staphylococcus simulans. The detectionlimit tested with purified genomic DNA from 5 strains of S. pneumoniaewas 1 to 10 genome copies. In addition, 5 quinolone-resistant and 2quinolone-sensitive clinical isolates of S. pneumoniae were tested tofurther validate the developed multiplex PCR coupled with capture probehybridization assays. There was a perfect correlation between detectionof S. pneumoniae gyrA and parC mutations and the susceptibility toquinolone.

The complete assay for the specific detection of S. pneumoniae and itssusceptibility to quinolone contains the S. pneumoniae-specific primers(SEQ ID NOs. 1179 and 1181) described in Example 20 and the multiplexcontaining the S. pneumoniae gyrA-specific and parC-specific primerpairs (SEQ ID NOS. 2040 and 2041 and SEQ ID NOs. 2044 and 2045).Amplification is coupled with post-PCR hybridization with the internalprobe specific to S. pneumoniae (SEQ ID NO. 1180) described in Exampleand the internal probes specific to each of the S. pneumoniae gyrA andparC variants (SEQ ID NOs. 2042, 2043 and 2046).

Example 36 Detection of Extended-Spectrum TEM-Type β-Lactamases inEscherichia coli

The analysis of TEM sequences which confer resistance tothird-generation cephalosporins and to β-lactamase inhibitors allowedthe identification of amino acid substitutions Met-69 to Ile or Leu orVal, Ser-130 to Gly, Arg-164 to Ser or H is, Gly-238 to Ser, Glu-240 toLys and Arg-244 to Ser or Cys or Thr or His or Leu. PCR primers SEQ IDNOs. 1907 and 1908 were designed to amplify TEM sequences. Internalprobes for the specific detection of wild-type TEM (SEQ ID NO. 2141) andfor each of the amino acid substitutions (SEQ ID NOs. 1909-1926)identified in TEM variants were designed to detect resistance tothird-generation cephalosporins and to β-lactamase inhibitors. Designand synthesis of primers and probes, and detection of the hybridizationwere performed as described in Example 7.

For all bacterial species, amplification was performed from purifiedgenomic DNA. One μl of genomic DNA at 0.1 ng/μl was transferred directlyto a 19 μl PCR mixture. Each PCR reaction contained 50 mM KCl, 10 mMTris-HCl (pH 9.0); 0.1% Triton X-100, 2.5 mM MgCl₂, 0.4 μM of theTEM-specific primers SEQ ID NOs. 1907 and 1908, 200 μM (each) of thefour deoxynucleoside triphosphates, 0.05 mM bovine serum albumin (BSA)and 0.5 U Taq polymerase (Promega) coupled with TaqStart™ antibody. PCRamplification and agarose gel analysis of the amplified products wereperformed as described in Example 28. The optimal cycling conditions formaximum sensitivity and specificity were 3 minutes at 95° C. for initialdenaturation, then forty cycles of three steps consisting of 5 secondsat 95° C., 30 seconds at 55° C. and 30 seconds at 72° C., followed byterminal extension at 72° C. for 2 minutes.

The specificity of the TEM-specific primers with 40-cycle PCR wasverified by using 0.1 ng of purified genomic from the followingbacteria: three third-generation cephalosporin-resistant Escherichiacoli strains (one with TEM-10, one with TEM-28 and the other withTEM-49), two third-generation cephalosporin-sensitive Escherichia colistrain (one with TEM-1 and the other without TEM), one third-generationcephalosporin-resistant Klebsiella pneumoniae strain (with TEM-47), andone β-lactamase-inhibitor-resistant Proteus mirabilis strain (withTEM-39). Amplification with the TEM-specific primers was detected onlyfor strains containing TEM.

The sensitivity of the assay with 40-cycle PCR was verified with threeE. coli strains containing TEM-1 or TEM-10 or TEM-49, one K. pneumoniaestrain containing TEM-47 and one P. mirabilis strain containing TEM-39.The detection limit was 5 to 100 copies of genomic DNA, depending on theTEM-containing strains tested.

The TEM-specific primers SEQ ID NOs. 1907 and 1908 were used inmultiplex with the Escherichia coli/Shigella sp.-specific primers SEQ IDNOs. 1661 and 1665 described in Example 28 to allow the completeidentification of Escherichia coli/Shigella sp. and the susceptibilityto β-lactams. PCR amplification with 0.4 μM of each of the primers andagarose gel analysis of the amplified products was performed asdescribed above.

The specificity of the multiplex with 40-cycle PCR was verified by using0.1 ng of purified genomic DNA from the following bacteria: threethird-generation cephalosporin-resistant Escherichia coli strains (onewith TEM-10, one with TEM-28 and the other with TEM-49), twothird-generation cephalosporin-sensitive Escherichia coli strain (onewith TEM-1 and the other without TEM), one third-generationcephalosporin-resistant Klebsiella pneumoniae strain (with TEM-47), andone β-lactamase-inhibitor-resistant Proteus mirabilis strain (withTEM-39). The multiplex was highly specific to Escherichia coli strainscontaining TEM.

The complete assay for detection of TEM-type β-lactamases in E. coliincludes PCR amplification using the multiplex containing theTEM-specific primers (SEQ ID NOs. 1907 and 1908) and the Escherichiacoli/Shigella sp.-specific primers (SEQ ID NOs. 1661 and 1665) coupledwith post PCR-hybridization with the internal probes specific towild-type TEM (SEQ ID NO. 2141) and to the TEM variants (SEQ ID NOs.1909-1926).

Example 37 Detection of Extended-Spectrum SHV-Type β-Lactamases inKlebsiella pneumoniae

The comparison of SHV sequences, which confer resistance tothird-generation cephalosporins and to β-lactamase inhibitors, allowedthe identification of amino acid substitutions Ser-130 to Gly, Asp-179to Ala or Asn, Gly-238 to Ser, and Glu-240 to Lys. PCR primer pair SEQID NOs. 1884 and 1885 was designed to amplify SHV sequences. Internalprobes for the specific identification of wild-type SHV (SEQ ID NO.1896) and for each of the amino acid substitutions (SEQ ID NOs.1886-1895 and 1897-1898) identified in SHV variants were designed todetect resistance to third-generation cephalosporins and to β-lactamaseinhibitors. Design and synthesis of primers and probes, and detection ofthe hybridization were performed as described in Example 7.

For all bacterial species, amplification was performed from purifiedgenomic DNA. One μl of genomic DNA at 0.1 ng/μl was transferred directlyto a 19 μl PCR mixture. Each PCR reaction contained 50 mM KCl, 10 mMTris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM MgCl₂, 0.4 μM of theSHV-specific primers SEQ ID NO. 1884 and 1885, 200 μM (each) of the fourdeoxynucleoside triphosphates, 0.05 mM bovine serum albumin (BSA) and0.5 U Taq polymerase (Promega) coupled with TaqStart™ antibody. PCRamplification and agarose gel analysis of the amplified products wereperformed as described in Example 28. The optimal cycling conditions formaximum sensitivity and specificity were 3 minutes at 95° C. for initialdenaturation, then forty cycles of three steps consisting of 5 secondsat 95° C., 30 seconds at 55° C. and 30 seconds at 72° C., followed byterminal extension at 72° C. for 2 minutes.

The specificity of the SHV-specific primers with 40-cycle PCR wasverified by using 0.1 ng of purified genomic from the followingbacteria: two third-generation cephalosporin-resistant Klebsiellapneumoniae strains (one with SHV-2a and the other with SHV-12), onethird-generation cephalosporin-sensitive Klebsiella pneumoniae strain(with SHV-1), two third-generation cephalosporin-resistant Escherichiacoli strains (one with SHV-8 and the other with SHV-7), and twothird-generation cephalosporin-sensitive Escherichia coli strains (onewith SHV-1 and the other without any SHV). Amplification with theSHV-specific primers was detected only for strains containing SHV.

The sensitivity of the assay with 40-cycle PCR was verified with fourstrains containing SHV. The detection limit was 10 to 100 copies ofgenomic DNA, depending on the SHV-containing strains tested.

The amplification was coupled with post-PCR hybridization with theinternal probes specific for identification of wild-type SHV (SEQ ID NO.1896) and for each of the amino acid substitutions (SEQ ID NOs.1886-1895 and 1897-1898) identified in SHV variants. The specificity ofthe probes was verified with six strains containing various SHV enzymes,one Klebsiella pneumoniae strain containing SHV-1, one Klebsiellapneumoniae strain containing SHV-2a, one Klebsiella pneumoniae straincontaining SHV-12, one Escherichia coli strain containing SHV-1, oneEscherichia coli strain containing SHV-7 and one Escherichia coli straincontaining SHV-8. The probes correctly detected each of the SHV genesand their specific mutations. There was a perfect correlation betweenthe SHV genotype of the strains and the susceptibility to β-lactamantibiotics.

The SHV-specific primers SEQ ID NOs. 1884 and 1885 were used inmultiplex with the K. pneumoniae-specific primers SEQ ID NOs. 1331 and1332 described in Example 29 to allow the complete identification of K.pneumoniae and the susceptibility to β-lactams. PCR amplification with0.4 μM of each of the primers and agarose gel analysis of the amplifiedproducts were performed as described above.

The specificity of the multiplex with 40-cycle PCR was verified by using0.1 ng of purified genomic DNA from the following bacteria: three K.pneumoniae strains containing SHV-1, one Klebsiella pneumoniae straincontaining SHV-2a, one Klebsiella pneumoniae strain containing SHV-12,one K rhinoscleromatis strain containing SHV-1, one Escherichia colistrain without SHV. The multiplex was highly specific to Klebsiellapneumoniae strain containing SHV.

Example 38 Development of a PCR Assay for the Detection andIdentification of Neisseria gonorrhoeae and its Associated TetracyclineResistance Gene tetM

The analysis of publicly available tetM sequences revealed conservedregions allowing the design of PCR primers specific to tetM sequences.The PCR primer pair SEQ ID NOs. 1588 and 1589 was used in multiplex withthe Neisseria gonorrhoeae-specific primers SEQ ID NOs. 551 and 552described in Example 31. Sequence alignment analysis of tetM sequencesrevealed regions suitable for the design of an internal probe specificto tetM (SEQ ID NO. 2254). PCR amplification was carried out on PTC-200thermocyclers (MJ Research) using 0.4 μM of each primer pair asdescribed in Example 28. The optimal cycling conditions for maximumsensitivity and specificity were as follow: three minutes at 95° C. forinitial denaturation, then forty cycles of two steps consisting of 1second at 95° C. and 30 seconds at 60° C., followed by terminalextension at 72° C. for 2 minutes.

The specificity of the multiplex PCR assay with 40-cycle PCR wasverified by using 0.1 ng of purified genomic DNA from the followingbacteria: two tetracycline-resistant Escherichia coli strains (onecontaining the tetracycline-resistant gene tetB and the other containingthe tetracycline-resistant gene tetC), one tetracycline-resistantPseudomonas aeruginosa strain (containing the tetracycline-resistantgene tetA), nine tetracycline-resistant Neisseria gonorrhoeae strains,two tetracycline-sensitive Neisseria meningitidis strains, onetetracycline-sensitive Neisseria polysaccharea strain, onetetracycline-sensitive Neisseria sicca strain and onetetracycline-sensitive Neisseria subflava strain. Amplification withboth the tetM-specific and Neisseria gonorrhoeae-specific primers wasdetected only for N. gonorrhoeae strains containing tetM. There was aweak amplification signal using Neisseria gonorrhoeae-specific primersfor the following species: Neisseria sicca, Neisseria polysaccharea andNeisseria meningitidis. There was a perfect correlation between the tetMgenotype and the tetracycline susceptibility pattern of the Neisseriagonorrhoeae strains tested. The internal probe specific to N.gonorrhoeae SEQ ID NO. 2166 described in Example 31 can discriminateNeisseria gonorrhoeae from the other Neisseria sp.

The sensitivity of the assay with 40-cycle PCR was verified with twotetracycline resistant strains of N. gonorrhoeae. The detection limitwas 5 copies of genomic DNA for both strains.

Example 39 Development of a PCR Assay for the Detection andIdentification of Shigella Sp. and Their Associated TrimethoprimResistance Gene dhfrIa

The analysis of publicly available dhfrIa and other dhfr sequencesrevealed regions allowing the design of PCR primers specific to dhfrIasequences. The PCR primer pair (SEQ ID NOs. 1459 and 1460) was used inmultiplex with the Escherichia coli/Shigella sp.-specific primers SEQ IDNOs. 1661 and 1665 described in Example 28. Sequence alignment analysisof dhfrIa sequences revealed regions suitable for the design of aninternal probe specific to dhfrIa (SEQ ID NO. 2253). PCR amplificationand agarose gel analysis of the amplified products were performed asdescribed in Example 28 with an annealing temperature of 60° C. Thespecificity of the multiplex assay with 40-cycle PCR was verified byusing 0.1 ng of purified genomic DNA from a panel of bacteria. The listincluded the following trimethoprim-sensitive strains, Salmonellatyphimyurium, Salmonella typhi, Salmonella enteritidis, Tatumellaptyseos, Klebsiella pneumoniae, Enterobacter aerogenes, Citrobacterfarmeri, Campylobacter jejuni, Serratia marcescens, Shigelladysenteriae, Shigella flexneri, Shigella sonnei, sixtrimethoprim-resistant Escherichia coli strains (containing dhfrIa ordhfrV or dhfrVII or dhfrXII or dhfrXIII or dhfrXV), fourtrimethoprim-resistant strains containing dhfrIa (Shigella sonnei,Shigella flexneri, Shigella dysenteriae and Escherichia coli). There wasa perfect correlation between the dhfrIa genotype and the trimethoprimsusceptibility pattern of the Escherichia coli and Shigella sp. strainstested. The dhfrIa primers were specific to the dhfrIa gene and did notamplify any of the other trimethoprim-resistant dhfr genes tested. Thesensitivity of the multiplex assay with 40-cycle PCR was verified withthree strains of trimethoprim-resistant strains of Shigella sp. Thedetection limit was 5 to 10 genome copies of DNA, depending on theShigella sp. strains tested.

Example 40 Development of a PCR Assay for the Detection andIdentification of Acinetobacter baumannii and its AssociatedAminoglycoside Resistance Gene aph(3′)-VIa

The comparison of publicly available aph(3′)-VIa sequence revealedregions allowing the design of PCR primers specific to aph(3′)-VIa. ThePCR primer pair (SEQ ID NOs. 1404 and 1405) was used in multiplex withthe Acinetobacter baumannii-specific primers SEQ ID NOs. 1692 and 1693described in Example 30. Analysis of the aph(3′)-VIa sequence revealedregion suitable for the design of an internal probe specific toaph(3′)-VIa (SEQ ID NO. 2252). PCR amplification and agarose gelanalysis of the amplified products were performed as described inExample 28. The specificity of the multiplex assay with 40-cycle PCR wasverified by using 0.1 ng of purified genomic DNA from a panel ofbacteria including: two aminoglycoside-resistant A. baumanni strains(containing aph(3′)-VIa), one aminoglycoside-sensitive A. baumanistrain, one of each of the following aminoglycoside-resistant bacteria,one Serratia marcescens strain containing the aminoglycoside-resistantgene aacC1, one Serratia marcescens strain containing theaminoglycoside-resistant gene aacC4, one Enterobacter cloacae straincontaining the aminoglycoside-resistant gene aacC2, one Enterococcusfaecalis containing the aminoglycoside-resistant gene aacA-aphD, onePseudomonas aeruginosa strain containing the aminoglycoside-resistantgene aac6IIa and one of each of the following aminoglycoside-sensitivebacterial species, Acinetobacter anitratus, Acinetobacter lwoffi,Psychobbacter phenylpyruvian, Neisseria gonorrhoeae, Haemophilushaemolyticus, Haemophilus influenzae, Yersinia enterolitica, Proteusvulgaris, Eikenella corrodens, Escherichia coli. There was a perfectcorrelation between the aph(3′)-VIa genotype and the aminoglycosidesusuceptibility pattern of the A. baumannii strains tested. Theaph(3′)-VIa-specific primers were specific to the aph(3′)-VIa gene anddid not amplify any of the other aminoglycoside-resistant genes tested.The sensitivity of the multiplex assay with 40-cycle PCR was verifiedwith two strains of aminoglycoside-resistant strains of A. baumannii.The detection limit was 5 genome copies of DNA for both A. baumanniistrains tested.

Example 41 Specific Identification of Bacteroides fragilis Using atpD(V-Type) Sequences

The comparison of atpD (V-type) sequences from a variety of bacterialspecies allowed the selection of PCR primers for Bacteroides fragilis.The strategy used to design the PCR primers was based on the analysis ofa multiple sequence alignement of various atpD sequences from B.fragilis, as well as atpD sequences from the related species B. dispar,bacterial genera and archaea, especially representatives withphylogenetically related atpD sequences. A careful analysis of thisalignment allowed the selection of oligonucleotide sequences which areconserved within the target species but which discriminate sequencesfrom other species, especially from closely related species B. dispar,thereby permitting the species-specific and ubiquitous detection andidentification of the target bacterial species.

The chosen primer pair, SEQ ID NOs. 2134-2135, produces an amplificationproduct of 231 bp. Standard PCR was carried out on PTC-200 thermocyclers(MJ Research Inc.) using 0.4 μM of each primers pair as described inExample 28. The optimal cycling conditions for maximum sensitivity andspecificity were as follows: three minutes at 95° C. for initialdenaturation, then forty cycles of two steps consisting of 1 second at95° C. and 30 seconds at 60° C., followed by terminal extension at 72°C. for 2 minutes.

The format of this assay is not limited to the one described above. Aperson skilled in the art could adapt the assay for different formatssuch as PCR with real-time detection using molecular beacon probes.Molecular beacon probes designed to be used in this assay include, butare not limited to, SEQ ID NO. 2136 for the detection of the B. fragilisamplicon.

Example 42 Evidence for Horizontal Gene Transfer in the Evolution of theElongation Factor Tu in Enterococci

Overview

The elongation factor Tu, encoded by tuf genes, is a GTP binding proteinthat plays a central role in protein synthesis. One to three tuf genesper genome are present depending on the bacterial species. Most low G+Cgram-positive bacteria carry only one tuf gene. We have designeddegenerate PCR primers derived from consensus sequences of the tuf geneto amplify partial tuf sequences from 17 enterococcal species and otherphylogenetically related species. The amplified DNA fragments weresequenced either by direct sequencing or by sequencing cloned insertscontaining putative amplicons. Two different tuf genes (tufA and tufB)were found in 11 enterococcal species, including Enterococcus avium, E.casseliflavus, E. dispar, E. durans, E. faecium, E. gallinarum, E.hirae, E. malodoratus, E. mundtii, E. pseudoavium, and E. raffinosus.For the other six enterococcal species (E. cecorum, E. columbae, E.faecalis, E. sulfureus, E. saccharolyticus, and E. solitarius), only thetufA gene was present. Based on 16S rRNA gene sequence analysis, the 11species having two tuf genes all share a common ancestor, while the sixspecies having only one copy diverged from the enterococcal lineagebefore that common ancestor. The presence of one or two copies of thetuf gene in enterococci was confirmed by Southern hybridization.Phylogenetic analysis of tuf sequences demonstrated that theenterococcal tufA gene branches with the Bacillus, Listeria andStaphylococcus genera, while the enterococcal tufB gene clusters withthe genera Streptococcus and Lactococcus. Primary structure analysisshowed that four amino acid residues within the sequenced regions areconserved and unique to the enterococcal tufB genes and the tuf genes ofstreptococci and L. lactis. The data suggest that an ancestralstreptococcus or a streptococcus-related species may have horizontallytransferred a tuf gene to the common ancestor of the 11 enterococcalspecies which now carry two tuf genes.

Introduction

The elongation factor Tu (EF-Tu) is a GTP binding protein playing acentral role in protein synthesis. It mediates the recognition andtransport of aminoacyl-tRNAs and their positioning to the A-site of theribosome. The highly conserved function and ubiquitous distributionrender the elongation factor a valuable phylogenetic marker amongeubacteria and even throughout the archaebacterial and eukaryotickingdoms. The tuf genes encoding elongation factor Tu are present invarious copy numbers per bacterial genome. Most gram-negative bacteriacontain two tuf genes. As found in Escherichia coli, the two genes,while being almost identical in sequence, are located in different partsof the bacterial chromosome. However, recently completed microbialgenomes revealed that only one tuf gene is found in Helicobacter pylorias well as in some obligate parasitic bacteria, such as Borreliaburgdorferi, Rickettsia prowazekii, and Treponema pallidum, and in somecyanobacteria. In most gram-positive bacteria studied so far, only onetuf gene was found. However, Southern hybridization showed that thereare two tuf genes in some clostridia as well as in Streptomycescoelicolor and S. lividans. Up to three tuf-like genes have beenidentified in S. ramocissimus.

Although massive prokaryotic gene transfer is suggested to be one of thefactors responsible for the evolution of bacterial genomes, the genesencoding components of the translation machinery are thought to behighly conserved and difficult to be transferred horizontally due to thecomplexity of their interactions. However, a few recent studiesdemonstrated evidence that horizontal gene transfer has also occurred inthe evolution of some genes coding for the translation apparatus,namely, 16S rRNA and some aminoacyl-tRNA synthetases. No further datasuggest that such a mechanism is involved in the evolution of theelongation factors. Previous studies concluded that the two copies oftuf genes in the genomes of some bacteria resulted from an ancient eventof gene duplication. Moreover, a study of the tuf gene in R. prowazekiisuggested that intrachromosomal recombination has taken place in theevolution of the genome of this organism.

To date, little is known about the tuf genes of enterococcal species. Inthis study, we analyzed partial sequences of tuf genes in 17enterococcal species, namely, E. avium, E. casseliflavus, E. cecorum, E.columbae, E. dispar, E. durans, E. faecalis, E. faecium, E. gallinarum,E. hirae, E. malodoratus, E. mundtii, E. pseudoavium, E. raffinosus, E.saccharolyticus, E. solitarius, and E. sulfureus. We report here thepresence of two divergent copies of tuf genes in 11 of theseenterococcal species. The 6 other species carried a single tuf gene. Theevolutionary implications are discussed.

Materials and Methods

Bacterial strains. Seventeen enterococcal strains and othergram-positive bacterial strains obtained from the American Type CultureCollection (ATCC, Manassas, Va.) were used in this study (Table 16). Allstrains were grown on sheep blood agar or in brain-heart infusion brothprior to DNA isolation.

DNA isolation. Bacterial DNAs were prepared using the G NOME DNAextraction kit (Bio101, Vista, Calif.) as previously described.

Sequencing of putative tuf genes. In order to obtain the tuf genesequences of enterococci and other gram-positive bacteria, twosequencing approaches were used: 1) sequencing of cloned PCR productsand 2) direct sequencing of PCR products. A pair of degenerate primers(SEQ ID NOs. 664 and 697) were used to amplify an 886-bp portion of thetuf genes from enterococcal species and other gram-positive bacteria aspreviously described. For E. avium, E. casseliflavus, E. dispar, E.durans, E. faecium, E. gallinarum, E. hirae, E. mundtii, E. pseudoavium,and E. raffinosus, the amplicons were cloned using the Original TAcloning kit (Invitrogen, Carlsbad, Calif.) as previously described. Fiveclones for each species were selected for sequencing. For E. cecorum, E.faecalis, E. saccharolyticus, and E. solitarius as well as the othergram-positive bacteria, the sequences of the 886-bp amplicons wereobtained by direct sequencing. Based on the results obtained from theearlier rounds of sequencing, two pairs of primers were designed forobtaining the partial tuf sequences from the other enterococcal speciesby direct sequencing. One pair of primers (SEQ ID NOs. 543 and 660) wereused to amplify the enterococcal tuf gene fragments from E. columbae, E.malodoratus, and E. sulfureus. Another pair of primers (SEQ ID NOs. 664and 661) were used to amplify the second tuf gene fragments from E.avium, E. malodoratus, and E. pseudoavium.

Prior to direct sequencing, PCR products were electrophoresed on 1%agarose gel at 120V for 2 hours. The gel was then stained with 0.02%methylene blue for 30 minutes and washed twice with autoclaved distilledwater for 15 minutes. The gel slices containing PCR products of theexpected sizes were cut out and purified with the QIAquick gelextraction kit (QIAgen Inc., Mississauga, Ontario, Canada) according tothe manufacturer's instructions. PCR mixtures for sequencing wereprepared as described previously. DNA sequencing was carried out withthe Big Dye™ Terminator Ready Reaction cycle sequencing kit using a 377DNA sequencer (PE Applied Biosystems, Foster City, Calif.). Both strandsof the amplified DNA were sequenced. The sequence data were verifiedusing the Sequencer™ 3.0 software (Gene Codes Corp., Ann Arbor, Mich.).

Sequence analysis and phylogenetic study. Nucleotide sequences of thetuf genes and their respective flanking regions for E. faecalis,Staphylococcus aureus, and Streptococcus pneumoniae, were retrieved fromthe TIGR microbial genome database and S. pyogenes from the Universityof Oklahoma database. DNA sequences and deduced protein sequencesobtained in this study were compared with those in all publiclyavailable databases using the BLAST and FASTA programs. Unlessspecified, sequence analysis was conducted with the programs from GCGpackage (Version 10; Genetics Computer Group, Madison, Wisc.). Sequencealignment of the tuf genes from 74 species representing all threekingdoms of life (Tables 16 and 17) were carried out by use of Pileupand corrected upon visual analysis. The N- and C-termini extremities ofthe sequences were trimmed to yield a common block of 201 amino acidssequences and equivocal residues were removed. Phylogenetic analysis wasperformed with the aid of PAUP 4.0b4 written by Dr. David L. Swofford(Sinauer Associates, Inc., Publishers, Sunderland, Mass.). The distancematrix and maximum parsimony were used to generate phylogenetic treesand bootstrap resampling procedures were performed using 500 and 100replications in each analysis, respectively.

Protein structure analysis. The crystal structures of (i)Thermusaquaticus EF-Tu in complex with Phe-tRNA^(Phe) and a GTP analog and (ii)E. coli EF-Tu in complex with GDP served as templates for constructingthe equivalent models for enterococcal EF-Tu. Homology modeling ofprotein structure was performed using the SWISS-MODEL server andinspected using the SWISS-PDB viewer version 3.1.

Southern hybridization. In a previous study, we amplified and cloned an803-bp PCR product of the tuf gene fragment from E. faecium. Twodivergent sequences of the inserts, which we assumed to be tufA and tufBgenes, were obtained. The recombinant plasmid carrying either tufA ortufB sequence was used to generate two probes labeled with Digoxigenin(DIG)-11-dUTP by PCR incorporation following the instructions of themanufacturer (Boehringer Mannheim, Laval, Québec, Canada). Enterococcalgenomic DNA samples (1-2 μg) were digested to completion withrestriction endonucleases BglII and XbaI as recommended by the supplier(Amersham Pharmacia Biotech, Mississauga, Ontario, Canada). Theserestriction enzymes were chosen because no restriction sites wereobserved within the amplified tuf gene fragments of most enterococci.Southern blotting and filter hybridization were performed usingpositively charged nylon membranes (Boehringer Mannheim) and QuikHybhybridization solution (Stratagene Cloning Systems, La Jolla, Calif.)according to the manufacturers' instructions with modifications. Twentyμl of each digestion were electrophoresed for 2 h at 120V on a 0.8%agarose gel. The DNA fragments were denatured with 0.5 M NaOH andtransferred by Southern blotting onto a positively charged nylonmembrane (Boehringer Mannheim). The filters were pre-hybridized for 15min and then hybridized for 2 h in the QuikHyb solution at 68° C. witheither DIG-labeled probe. Posthybridization washings were performedtwice with 0.5×SSC, 1% SDS at room temperature for 15 min and twice inthe same solution at 60° C. for 15 min. Detection of bound probes wasachieved using disodium 3-(4-methoxyspiro(1,2-dioxetane-3,2′-(5′-chloro)tricyclo(3,3.1.1^(3.7))decan)-4-yl)phenyl phosphate (CSPD) (Boehringer Mannheim) as specifiedby the manufacturer.

GenBank submission. The GenBank accession numbers for partial tuf genesequences generated in this study are given in Table 16.

Results

Sequencing and nucleotide sequence analysis. In this study, allgram-positive bacteria other than enterococci yielded a single tufsequence of 886 bp using primers SEQ ID NOs. 664 and 697 (Table 16).Each of four enterococcal species including E. cecorum, E. faecalis, E.saccharolyticus, and E. solitarius also yielded one 886-bp tuf sequence.On the other hand, for E. avium, E. casseliflavus, E. dispar, E. durans,E. faecium, E. gallinarum, E. hirae, E. mundtii, E. pseudoavium, and E.raffinosus, direct sequencing of the 886-bp fragments revealedoverlapping peaks according to their sequence chromatograms, suggestingthe presence of additional copies of the tuf gene. Therefore, the tufgene fragments of these 10 species were cloned first and then sequenced.Sequencing data revealed that two different types of tuf sequences (tufAand tufB) are found in eight of these species including E.casseliflavus, E. dispar, E. durans, E. faecium, E. gallinarum, E.hirae, E. mundtii, and E. raffinosus. Five clones from E. avium and E.pseudoavium yielded only a single tuf sequence. These new sequence dataallowed the design of new primers specific for the enterococcal tufA ortufB sequences. Primers SEQ ID NOs. 543 and 660 were designed to amplifyonly enterococcal tufA sequences and a 694-bp fragment was amplifiedfrom all 17 enterococcal species. The 694-bp sequences of tufA genesfrom E. columbae, E. malodoratus, and E. sulfureus were obtained bydirect sequencing using these primers. Primers SEQ ID NOs. 664 and 661were designed for the amplification of 730-bp portion of tufB genes andyielded the expected fragments from 11 enterococcal species, includingE. malodoratus and the 10 enterococcal species in which heterogeneoustuf sequences were initially found. The sequences of the tufB fragmentsfor E. avium, E. malodoratus and E. pseudoavium were determined bydirect sequencing using the primers SEQ ID NOs. 664 and 661. Overall,tufA gene fragments were obtained from all 17 enterococcal species buttufB gene fragments were obtained with only 11 enterococcal species(Table 16).

The identities between tufA and tufB for each enterococcal species were68-79% at the nucleotide level and 81 to 89% at the amino acid level.The tufA gene is highly conserved among all enterococcal species withidentities varying from 87% to 99% for DNA and 93% to 99% for amino acidsequences, while the identities among tufB genes of enterococci variesfrom 77% to 92% for DNA and 91% to 99% for amino acid sequences,indicating their different origins and evolution (Table 18). Since E.solitarius has been transferred to the genus Tetragenococcus, which isalso a low G+C gram-positive bacterium, our sequence comparison did notinclude this species as an enterococcus. G+C content of enterococcaltufA sequences ranged from 40.8% to 43.1%, while that of enterococcaltufB sequences varied from 37.8% to 46.3%. Based on amino acid sequencecomparison, the enterococcal tufA gene products share higher identitieswith those of Abiotrophia adiacens, Bacillus subtilis, Listeriamonocytogenes, S. aureus, and S. epidermidis. On the other hand, theenterococcal tufB gene products share higher percentages of amino acididentity with the tuf genes of S. pneumoniae, S. pyogenes andLactococcus lactis (Table 18).

In order to elucidate whether the two enterococcal tuf sequences encodegenuine EF-Tu, the deduced amino acid sequences of both genes werealigned with other EF-Tu sequences available in SWISSPROT (Release 38).Sequence alignment demonstrated that both gene products are highlyconserved and carry all conserved residues present in this portion ofprokaryotic EF-Tu (FIG. 4). Therefore, it appears that both geneproducts could fulfill the function of EF-Tu. The partial tuf genesequences encode the portion of EF-Tu from residues 117 to 317, numberedas in E. coli. This portion makes up of the last four α-helices and twoβ-strands of domain I, the entire domain II and the N-terminal part ofdomain III on the basis of the determined structures of E. coli EF-Tu.

Based on the deduced amino acid sequences, the enterococcal tufB geneshave unique conserved residues Lys129, Leu140, Ser230, and Asp234 (E.coli numbering) that are also conserved in streptococci and L. lactis,but not in the other bacteria (FIG. 4). All these residues are locatedin loops except for Ser230. In other bacteria the residue Ser230 issubstituted for highly conserved Thr, which is the 5^(th) residue of thethird β-strand of domain II. This region is partially responsible forthe interaction between the EF-Tu and aminoacyl-tRNA by the formation ofa deep pocket for any of the 20 naturally occurring amino acids.According to our three-dimensional model (data not illustrated), thesubstitution Thr230→Ser in domain II of EF-Tu may have little impact onthe capability of the pocket to accommodate any amino acid. However, thehigh conservation of Thr230 comparing to the unique Ser substitutionfound only in streptococci and 11 enterococci could suggest a subtlefunctional role for this residue.

The tuf gene sequences obtained for E. faecalis, S. aureus, S.pneumoniae and S. pyogenes were compared with their respectiveincomplete genome sequence. Contigs with more than 99% identity wereidentified. Analysis of the E. faecalis genome data revealed that thesingle E. faecalis tuf gene is located within an str operon where tuf ispreceded by fus that encodes the elongation factor G. This str operon ispresent in S. aureus and B. subtilis but not in the two streptococcalgenomes examined. The 700-bp or so sequence upstream the S. pneumoniaetuf gene has no homology with any known gene sequences. In S. pyogenes,the gene upstream of tuf is similar to a cell division gene, ftsW,suggesting that the tuf genes in streptococci are not arranged in a stroperon.

Phylogenetic analysis. Phylogenetic analysis of the tuf amino acidsequences with representatives of eubacteria, archeabacteria, andeukaryotes using neighbor-joining and maximum parsimony methods showedthree major clusters representing the three kingdoms of life. Bothmethods gave similar topologies consistent with the rRNA gene data (datanot shown). Within the bacterial Glade, the tree is polyphyletic buttufA genes from all enterococcal species always clustered with thosefrom other low G+C gram-positive bacteria (except for streptococci andlactococci), while the tufB genes of the 11 enterococcal species form adistinct cluster with streptococci and L. lactis (FIG. 5). Duplicatedgenes from the same organism do not cluster together, thereby notsuggesting evolution by recent gene duplication.

Southern hybridization. Southern hybridization of BglII/XbaI digestedgenomic DNA from 12 enterococcal species tested with the tufA probe(DIG-labeled tufA fragment from E. faecium) yielded two bands ofdifferent sizes in 9 species, which also carried two divergent tufsequences according to their sequencing data. For E. faecalis and E.solitarius, a single band was observed indicating that one tuf gene ispresent (FIG. 6). A single band was also found when digested genomic DNAfrom S. aureus, S. pneumoniae, and S. pyogenes were hybridized with thetufA probe (data not shown). For E. faecium, the presence of three bandscan be explained by the existence of a XbaI restriction site in themiddle of the tufA sequence, which was confirmed by sequencing data.Hybridization with the tufB probe (DIG-labeled tufB fragment of E.faecium) showed a banding profile similar to the one obtained with thetufA probe (data not shown).

Discussion

In this study, we have shown that two divergent copies of genes encodingthe elongation factor Tu are present in some enterococcal species.Sequence data revealed that both genes are highly conserved at the aminoacid level. One copy (tufA) is present in all enterococcal species,while the other (tufB) is present only in 11 of the 17 enterococcalspecies studied. Based on 16S rRNA sequence analysis, these 11 speciesare members of three different enterococcal subgroups (E. avium, E.faecium, and E. gallinarum species groups) and a distinct species (E.dispar). Moreover, 16S rDNA phylogeny suggests that these 11 speciespossessing 2 tuf genes all share a common ancestor before they furtherevolved to become the modern species. Since the six other species havingonly one copy diverged from the enterococcal lineage before that commonancestor, it appears that the presence of one tuf gene in these sixspecies is not attributable to gene loss.

Two clusters of low G+C gram-positive bacteria were observed in thephylogenetic tree of the tuf genes: one contains a majority of low G+Cgram-positive bacteria and the other contains lactococci andstreptococci. This is similar to the finding on the basis ofphylogenetic analysis of the 16S rRNA gene and the hrcA gene coding fora unique heat-shock regulatory protein. The enterococcal tufA genesbranched with most of the low G+C gram-positive bacteria, suggestingthat they originated from a common ancestor. On the other hand, theenterococcal tufB genes branched with the genera Streptococcus andLactococcus that form a distinct lineage separated from other low G+Cgram-positive bacteria (FIG. 5). The finding that these EF-Tu proteinsshare some conserved amino acid residues unique to this branch alsosupports the idea that they may share a common ancestor. Although theseconserved residues might result from convergent evolution upon aspecialized function, such convergence at the sequence level, even for afew residues, seems to be rare, making it an unlikely event. Moreover,no currently known selective pressure, if any, would account for keepingone versus two tuf genes in bacteria. The G+C contents of enterococcaltufA and tufB sequences are similar, indicating that they bothoriginated from low G+C gram-positive bacteria, in accordance with thephylogenetic analysis.

The tuf genes are present in various copy numbers in different bacteria.Furthermore, the two tuf genes are normally associated withcharacteristic flanking genes. The two tuf gene copies commonlyencountered within gram-negative bacteria are part of the bacterial stroperon and tRNA-tufB operon, respectively. The arrangement of tufA inthe str operon was also found in a variety of bacteria, includingThermotoga maritima, the most ancient bacteria sequenced so far, Aquifexaeolicus, cyanobacteria, Bacillus sp., Micrococcus luteus, Mycobacteriumtuberculosis, and Streptomyces sp. Furthermore, the tRNA-tufB operon hasalso been identified in Aquifex aeolicus, Thermus thermophilus, andChlamydia trachomatis. The two widespread tuf gene arrangements argue infavor of their ancient origins. It is noteworthy that most obligateintracellular parasites, such as Mycoplasma sp., R. prowazekii, B.burgdorferi, and T. pallidum, contain only one tuf gene. Their flankingsequences are distinct from the two conserved patterns as a result ofselection for effective propagation by an extensive reduction in genomesize by intragenomic recombination and rearrangement.

Most gram-positive bacteria with low G+C content sequenced to datecontain only a single copy of the tuf gene as a part of the str operon.This is the case for B. subtilis, S. aureus and E. faecalis. PCRamplification using a primer targeting a conserved region of the fusgene and the tufA-specific primer SEQ ID NO. 660, but not thetufB-specific primer SEQ ID NO. 661, yielded the expected amplicons forall 17 enterococcal species tested, indicating the presence of thefus-tuf organization in all enterococci (data not shown). However, inthe genomes of S. pneumoniae and S. pyogenes, the sequences flanking thetuf genes varies although the tuf gene itself remains highly conserved.The enterococcal tufB genes are clustered with streptococci, but atpresent we do not have enough data to identify the genes flanking theenterococcal tufB genes. Furthermore, the functional role of theenterococcal tufB genes remains unknown. One can only postulate that thetwo divergent gene copies are expressed under different conditions.

The amino acid sequence identities between the enterococcal tufA andtufB genes are lower than either i) those between the enterococcal tufAand the tuf genes from other low G+C gram-positive bacteria(streptococci and lactococci excluded) or ii) those between theenterococcal tufB and streptococcal and lactococcal tuf genes. Thesefindings suggest that the enterococcal tufA genes share a commonancestor with other low G+C gram-positive bacteria via the simple schemeof vertical evolution, while the enterococcal tufB genes are moreclosely related to those of streptococci and lactococci. The facts thatsome enterococci possess an additional tuf gene and that the singlestreptococcal tuf gene is not clustered with other low G+C gram-positivebacteria cannot be explained by the mechanism of gene duplication orintrachromosomal recombination. According to sequence and phylogeneticanalysis, we propose that the presence of the additional copy of the tufgenes in 11 enterococcal species is due to horizontal gene transfer. Thecommon ancestor of the 11 enterococcal species now carrying tufB genesacquired a tuf gene from an ancestral streptococcus or astreptococcus-related species during enterococcal evolution through genetransfer before the diversification of modern enterococci. Further studyof the flanking regions of the gene may provide more clues for theorigin and function of this gene in enterococci.

Recent studies of genes and genomes have demonstrated that considerablehorizontal transfer occurred in the evolution of aminoacyl-tRNAsynthetases in all three kingdoms of life. The heterogeneity of 16S rRNAis also attributable to horizontal gene transfer in some bacteria, suchas Streptomyces, Thermomonospora chromogena and Mycobacterium celatum.In this study, we provide the first example in support of a likelyhorizontal transfer of the tuf gene encoding the elongation factor Tu.This may be an exception since stringent functional constraints do notallow for frequent horizontal transfer of the tuf gene as with othergenes. However, enterococcal tuf genes should not be the only suchexception as we have noticed that the phylogeny of Streptomyces tufgenes is equally or more complex than that of enterococci. For example,the three tuf-like genes in a high G+C gram-positive bacterium, S.ramocissimus, branched with the tuf genes of phylogenetically divergentgroups of bacteria (FIG. 5). Another example may be the tuf genes inclostridia, which represent a phylogenetically very broad range oforganisms and form a plethora of lines and groups of variouscomplexities and depths. Four species belonging to three differentclusters within the genus Clostridium have been shown by Southernhybridization to carry two copies of the tuf gene. Further sequence dataand phylogenetic analysis may help interpreting the evolution of theelongation factor Tu in these gram-positive bacteria. Since the tufgenes and 16S rRNA genes are often used for phylogenetic study, theexistence of duplicate genes originating from horizontal gene transfermay alter the phylogeny of microorganisms when the laterally acquiredcopy of the gene is used for such analysis. Hence, caution should betaken in interpreting phylogenetic data. In addition, the two tuf genesin enterococci have evolved separately and are distantly related to eachother phylogenetically. The enterococcal tufB genes are less conservedand unique to the 11 enterococcal species only. We previouslydemonstrated that the enterococcal tufA genes could serve as a target todevelop a DNA-based assay for identification of enterococci. Theenterococcal tufB genes would also be useful in identification of these11 enterococcal species.

Example 43 Elongation Factor Tu (Tuf) and the F-ATPase Beta-Subunit(atpD) as Phylogenetic Tools for Species of the FamilyEnterobacteriaceae SUMMARY

The phylogeny of enterobacterial species commonly found in clinicalsamples was analyzed by comparing partial sequences of their elongationfactor Tu (tuf) genes and their F-ATPase beta-subunit (atpD) genes. A884-bp fragment for tuf and a 884- or 871-bp fragment for atpD weresequenced for 88 strains of 72 species from 25 enterobacterial genera.The atpD sequence analysis revealed a specific indel to Pantoea andTatumella species showing for the first time a tight phylogeneticaffiliation between these two genera. Comprehensive tuf and atpDphylogenetic trees were constructed and are in agreement with eachother. Monophyletic genera are Yersinia, Pantoea, Edwardsiella, Cedecea,Salmonella, Serratia, Proteus, and Providencia. Analogous trees wereobtained based on available 16S rDNA sequences from databases. tuf andatpD phylogenies are in agreement with the 16S rDNA analysis despite thesmaller resolution power for the latter. In fact, distance comparisonsrevealed that tuf and atpD genes provide a better resolution for pairsof species belonging to the family Enterobacteriaceae. However, 16S rDNAdistances are better resolved for pairs of species belonging todifferent families. In conclusion, tuf and atpD conserved genes aresufficiently divergent to discriminate different species inside thefamily Enterobacteriaceae and offer potential for the development ofdiagnostic tests based on DNA to identify enterobacterial species.

Introduction

Members of the family Enterobacteriaceae are facultatively anaerobicgram-negative rods, catalase-positive and oxydase-positive (Brenner,1984). They are found in soil, water, plants, and in animals frominsects to man. Many enterobacteria are opportunistic pathogens. Infact, members of this family are responsible for about 50% of nosocomialinfections in the United States (Brenner, 1984). Therefore, this familyis of considerable clinical importance.

Major classification studies on the family Enterobacteriaceae are basedon phenotypic traits (Brenner et al., 1999; Brenner et al., 1980; Dickey& Zumoff, 1988; Farmer III et al., 1980; Farmer III et al., 1985b;Farmer III et al., 1985a) such as biochemical reactions andphysiological characteristics. However, phenotypically distinct strainsmay be closely related by genotypic criteria and may belong to the samegenospecies (Bercovier et al., 1980; Hartl & Dykhuizen, 1984). Also,phenotypically close strains (biogroups) may belong to differentgenospecies, like Klebsiella pneumoniae and Enterobacter aerogenes(Brenner, 1984) for example. Consequently, identification andclassification of certain species may be ambiguous with techniques basedon phenotypic tests (Janda et al., 1999; Kitch et al., 1994; Sharma etal., 1990).

More advances in the classification of members of the familyEnterobacteriaceae have come from DNA-DNA hybridization studies (Brenneret al., 1993; Brenner et al., 1986; Brenner, et al., 1980; Farmer III,et al., 1980; Farmer III, et al., 1985b; Izard et al., 1981; Steigerwaltet al., 1976). Furthermore, the phylogenetic significance of bacterialclassification based on 16S rDNA sequences has been recognized by manyworkers (Stackebrandt & Goebel, 1994; Wayne et al., 1987). However,members of the family Enterobacteriaceae have not been subjected toextensive phylogenetic analysis of 16S rDNA (Sproer et al., 1999). Infact, this molecule was not thought to solve taxonomic problemsconcerning closely related species because of its very high degree ofconservation (Brenner, 1992; Sproer, et al., 1999). Another drawback ofthe 16S rDNA gene is that it is found in several copies within thegenome (seven in Escherichia coli and Salmonella typhimurium) (Hill &Harnish, 1981). Due to sequence divergence between the gene copies,direct sequencing of PCR products is often not suitable to achieve arepresentative sequence (Cilia et al., 1996; Hill & Harnish, 1981).Other genes such as gap and ompA (Lawrence et al., 1991), rpoB (Molletet al., 1997), and infB (Hedegaard et al., 1999) were used to resolvethe phylogeny of enterobacteria. However, none of these studies coveredan extensive number of species.

tuf and atpD are the genes encoding the elongation factor Tu (EF-Tu) andthe F-ATPase beta-subunit, respectively. EF-Tu is involved in peptidechain formation (Ludwig et al., 1990). The two copies of the tuf gene(tufA and tufB) found in enterobacteria (Sela et al., 1989) share highidentity level (99%) in Salmonella typhimurium and in E. coli. Therecombination phenomenon could explain sequence homogenization betweenthe two copies (Abdulkarim & Hughes, 1996; Grunberg-Manago, 1996).F-ATPase is present on the plasma membranes of eubacteria (Nelson &Taiz, 1989). It functions mainly in ATP synthesis (Nelson & Taiz, 1989)and the beta-subunit contains the catalytic site of the enzyme. EF-Tuand F-ATPase are highly conserved throughout evolution and showsfunctional constancy (Amann et al., 1988; Ludwig, et al., 1990).Recently, phylogenies based on protein sequences from EF-Tu and F-ATPasebeta-subunit showed good agreement with each other and with the rDNAdata (Ludwig et al., 1993).

We elected to sequence 884-bp fragments of tuf and atpD from 88clinically relevant enterobacterial strains representing 72 species from25 genera. These sequences were used to create phylogenetic trees thatwere compared with 16S rDNA trees. These trees revealed good agreementwith each others and demonstrated the high resolution of tuf and atpDphylogenies at the species level.

Materials and Methods

Bacterial strains and genomic material. All bacterial strains used inthis study were obtained from the American Type Culture Collection(ATCC) or the Deutsche Sammlung von Mikroorganismen and ZellkulturenGmbH (DSMZ). These enterobacteria can all be recovered from clinicalspecimens, but not all are pathogens. Whenever possible, we choose typestrains. Identification of all strains was confirmed by classicalbiochemical tests using the automated system MicroScan WalkAway-96system equipped with a Negative BP Combo Panel Type 15 (Dade BehringCanada). Genomic DNA was purified using the G NOME DNA kit (Bio 101).Genomic DNA from Yersinia pestis was kindly provided by Dr. Robert R.Brubaker. Strains used in this study and their descriptions are shown inTable 19.

PCR primers. The eubacterial tuf and atpD gene sequences available frompublic databases were analyzed using the GCG package (version 8.0)(Genetics Computer Group). Based on multiple sequence alignments, twohighly conserved regions were chosen for each genes, and PCR primerswere derived from these regions with the help of Oligo primer analysissoftware (version 5.0) (National Biosciences). A second 5′ primer wasdesign to amplify the gene atpD for few enterobacteria difficult toamplify with the first primer set. When required, the primers containedinosines or degeneracies to account for variable positions.Oligonucleotide primers were synthesized with a model 394 DNA/RNAsynthesizer (PE Applied Biosystems). PCR primers used in this study arelisted in Table 20.

DNA sequencing. An 884-bp portion of the tuf gene and an 884-bp portion(or alternatively an 871-bp portion for a few enterobacterial strains)of the atpD gene were sequenced for all enterobacteria listed in thefirst strain column of Table 19. Amplification was performed with 4 ngof genomic DNA. The 40-μl PCR mixtures used to generate PCR products forsequencing contained 1.0 μM each primer, 200 μM each deoxyribonucleosidetriphosphate (Pharmacia Biotech), 10 mM Tris-HCl (pH 9.0 at 25° C.), 50mM KCl, 0.1% (w/v) Triton X-100, 2.5 mM MgCl₂, 0.05 mM BSA, 0.3 U of TaqDNA polymerase (Promega) coupled with TaqStart™ antibody (ClontechLaboratories). The TaqStart™ neutralizing monoclonal antibody for TaqDNA polymerase was added to all PCR mixtures to enhance efficiency ofamplification (Kellogg et al., 1994). The PCR mixtures were subjected tothermal cycling (3 min at 95° C. and then 35 cycles of 1 min at 95° C.,1 min at 55° C. for tuf or 50° C. for atpD, and 1 min at 72° C., with a7-min final extension at 72° C.) using a PTC-200 DNA Engine thermocycler(MJ Research). PCR products having the predicted sizes were recoveredfrom an agarose gel stained for 15 min with 0.02% of methylene bluefollowed by washing in sterile distilled water for 15 min twice (Floreset al., 1992). Subsequently, PCR products having the predicted sizeswere recovered from gels using the QIAquick gel extraction kit (QIAGEN).

Both strands of the purified amplicons were sequenced using the ABIPrism BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE AppliedBiosystems) on an automated DNA sequencer (Model 377). Amplicons fromtwo independent PCR amplifications were sequenced for each strain toensure the absence of sequencing errors attributable to nucleotidemiscorporations by the Taq DNA polymerase. Sequence assembly wasperformed with the aid of Sequencher 3.0 software (Gene Codes).

Phylogenetic analysis. Multiple sequence alignments were performed usingPileUp from the GCG package (Version 10.0) (Genetics Computer Group) andchecked by eye with the editor SeqLab to edit sequences if necessary andto note which regions were to be excluded for phylogenetic analysis.Vibrio cholerae and Shewanella putrefaciens were used as outgroups.Bootstrap subsets (750 sets) and phylogenetic trees were generated withthe Neighbor Joining algorithm from Dr. David Swofford's PAUP(Phylogenetic Analysis Using Parsimony) Software version 4.0b4 (SinauerAssociates) and with tree-bisection branch-swapping. The distance modelused was Kimura (1980) two-parameter. Relative rate test was performedwith the aid of Phyltest program version 2.0 (c).

Results and Discussion

DNA Amplification, Sequencing And Sequence Alignments

A PCR product of the expected size of 884 bp was obtained for tuf and of884 or 871 bp for atpD from all bacterial strains tested. Aftersubtracting for biased primer regions and ambiguous single strand data,sequences of at least 721 bp for tuf and 713 bp for atpD were submittedto phylogenetic analyses. These sequences were aligned with tuf and atpDsequences available in databases to verify that the nucleotide sequencesindeed encoded a part of tested genes. Gaps were excluded to performphylogenetic analysis.

Signature Sequences

From the sequence alignments obtained from both tested genes, only oneinsertion was observed. This five amino acids insertion is locatedbetween the positions 325 and 326 of atpD gene of E. coli strain K-12(Saraste et al., 1981) and can be considered a signature sequence ofTatumella ptyseos and Pantoea species (FIG. 7). The presence of aconserved indel of defined length and sequence and flanked by conservedregions could suggest a common ancestor, particularly when members of agiven taxa share this indel (Gupta, 1998). To our knowledge, highrelatedness between the genera Tatumella and Pantoea is demonstrated forthe first time.

Enterobacter agglomerans ATCC 27989 sequence does not possess the fiveamino acid indel (FIG. 7). This indel could represent a useful marker tohelp resolve the Enterobacter agglomerans and Pantoea classification.Indeed, the transfer of Enterobacter agglomerans to Pantoea agglomeranswas proposed in 1989 bp Gavini et al. (Gavini et al., 1989). However,some strains are provisionally classified as Pantoea sp. until theirinterrelatedness is elucidated (Gavini, et al., 1989). Since thetransfer was proposed, the change of nomenclature has not yet been madefor all Enterobacter agglomerans in the ATCC database. The absence ofthe five amino acids indel suggests that some strains of Enterobacteragglomerans most likely do not belong to the genus Pantoea.

Phylogenetic Trees Based On Partial Tuf Sequences, atpD Sequences, andPublished 16S Rdna Data of Members of the Enterobacteriaceae.

Representative trees constructed from tuf and atpD sequences with theneighbor-joining method are shown in FIG. 8. The phylogenetic treesgenerated from partial tuf sequences and atpD sequences are verysimilar. Nevertheless, atpD tree shows more monophyletic groupscorresponding to species that belong to the same genus. These groups aremore consistent with the actual taxonomy. For both genes, some generaare not monophyletic. These results support previous phylogenies basedon the genes gap and ompA (Lawrence, et al., 1991), rpoB (Mollet, etal., 1997), and infB (Hedegaard, et al., 1999) which all showed that thegenera Escherichia and Klebsiella are polyphyletic. There were fewdifferences in branching between tuf and atpD genes.

Even though Pantoea agglomerans and Pantoea dispersa indels wereexcluded for phylogenetic analysis, these two species grouped togetherand were distant from Enterobacter agglomerans ATCC 27989, addinganother evidence that the latter species is heterogenous and that notall members of this species belong to the genus Pantoea. In fact, the E.agglomerans strain ATCC 27989 exhibits branch lengths similar to othersEnterobacter species with both genes. Therefore, we suggest that thisstrain belong to the genus Enterobacter until further reclassificationof that genus.

tuf and atpD trees exhibit very short genetic distances between taxabelonging to the same genetic species including species segregated forclinical considerations. This first concern E. coli and Shigella speciesthat were confirmed to be the same genetic species by hybridizationstudies (Brenner et al., 1972; Brenner et al., 1972; Brenner et al.,1982) and phylogenies based on 16S rDNA (Wang et al., 1997) and rpoBgenes (Mollet, et al., 1997). Hybridization studies (Bercovier, et al.,1980) and phylogeny based on 16S rDNA genes (Ibrahim et al., 1994)demonstrated also that Yersinia pestis and Y. pseudotuberculosis are thesame genetic species. Among Yersinia pestis and Y. pseudotuberculosis,the three Klebsiella pneumoniae subspecies, E. coli-Shigella species,and Salmonella choleraesuis subspecies, Salmonella is a less tightlyknit species than the other genetic species. The same is true for E.coli and Shigella species.

Escherichia fergusonii is very close to E. coli-Shigella geneticspecies. This observation is corroborated by 16S rDNA phylogeny(McLaughlin et al., 2000) but not by DNA hybridization values. In fact,E. fergusonii is only 49% to 63% related to E. coli-Shigella (FarmerIII, et al., 1985b). It was previously observed that very recentlydiverged species may not be recognizable based on 16S rDNA sequencesalthough DNA hybridization established them as different species (Fox etal., 1992). Therefore, E. fergusonii could be a new “quasi-species”.

atpD phylogeny revealed Salmonella subspecies divisions consistent withthe actual taxonomy. This result was already observed by Christensen etal. (Christensen & Olsen, 1998). Nevertheless, tuf partial sequencesdiscriminate less than atpD between Salmonella subspecies.

Overall, tuf and atpD phylogenies exhibit enough divergence betweenspecies to ensure efficient discrimination. Therefore, it could be easyto distinguish phenotypically close enterobacteria belonging todifferent genetic species such as Klebsiella pneumoniae and Enterobacteraerogenes.

Phylogenetic relationships between Salmonella, E. coli and C. freundiiare not well defined. 16S rDNA and 23S rDNA sequence data reveals acloser relationship between Salmonella and E. coli than betweenSalmonella and C. freundii (Christensen et al., 1998), while DNAhomology studies (Selander et al., 1996) and infB phylogeny (Hedegaard,et al., 1999) showed that Salmonella is more closely related to C.freundii than to E. coli. In that regard, tuf and atpD phylogenies arecoherent with 16S rDNA and 23S rDNA sequence analysis.

Phylogenetic analyses were also performed using amino acids sequences.tuf tree based on amino acids is characterized by a better resolutionbetween taxa outgroup and taxa ingroup (enterobacteria) than tree basedon nucleic acids whereas atpD trees based on amino acids and nucleicacids give almost the same resolution between taxa outgroup and ingroup(data not shown).

Relative rate test (or two cluster test (Takezaki et al., 1995))evaluates if evolution is constant between two taxa. Before to apply thetest, the topology of a tree is determined by tree-building methodwithout the assumption of rate constancy. Therefore, two taxa (or twogroups of taxa) are compared with a third taxon that is an outgroup ofthe first two taxa (Takezaki, et al., 1995). Few pairs of taxa thatexhibited a great difference between their branch lengths at particularnodes were chosen to perform the test. This test reveals that tuf andatpD are not constant in their evolution within the familyEnterobacteriaceae. For tuf, for example, the hypothesis of rateconstancy is rejected (Z value higher than 1.96) between Yersiniaspecies. The same is true for Proteus species. For atpD, for example,evolution is not constant between Proteus species, between Proteusspecies and Providencia species, and between Yersinia species andEscherichia coli. For 16S rDNA, for example, evolution is not constantbetween two E. coli, between E. coli and Enterobacter aerogenes, andbetween E. coli and Proteus vulgaris. These results suggest that tuf,atpD and 16S rDNA could not serve as a molecular clock for the entirefamily Enterobacteriaceae.

Since the number and the nature of taxa can influence topology of trees,phylogenetic trees from tuf and atpD were reconstructed using sequencescorresponding to strains for which 16S rDNA genes were published inGenEMBL. These trees were similar to those generated using 16S rDNA(FIG. 9). Nevertheless, 16S rDNA tree gave poorer resolution power thantuf and atpD gene trees. Indeed, these latter exhibited lessmultifurcation (polytomy) than the 16S rDNA tree.

Comparison of Distances Based on tuf, atpD, and 16S rDNA Data.

tuf, atpD, and 16S rDNA distances (i.e. the number of differences pernucleotide site) were compared with each other for each pair of strains.We found that the tuf and atpD distances were respectively 2.268±0.965and 2.927±0.896 times larger than 16S rDNA distances (FIG. 10 a and b).atpD distances were 1.445±0.570 times larger than tuf distances (FIG. 10c). FIG. 10 also shows that the tuf, atpD, and 16S rDNA distancesbetween members of different species of the same genus (0.053±0.034,0.060±0.020, and 0.024±0.010, respectively) were in mean smaller thanthe distances between members of different genera belonging to the samefamily (0.103±0.053, 0.129±0.051, and 0.044±0.013, respectively).However, the overlap exhibits with standard deviations add to a focus ofevidences that some enterobacterial genera are not well defined(Brenner, 1984). In fact, many distances for pairs of species especiallybelonging to the genera Escherichia, Shigella, Enterobacter,Citrobacter, Klebsiella, and Kluyvera overlap distances for pairs ofspecies belonging to the same genus (FIG. 10). For example, distancesfor pairs composed by species of Citrobacter and species of Klebsiellaoverlap distances for pairs composed by two Citrobacter or by twoKlebsiella.

Observing the distance distributions, 16S rDNA distances reveal a clearseparation between the families Enterobacteriaceae and Vibrionaceaedespite the fact that the family Vibrionaceae is genetically very closeto the Enterobacteriaceae (FIG. 10 a and b). Nevertheless, tuf and atpDshow higher discriminating power below the family level (FIG. 10 a andb).

There were some discrepancies in the relative distances for the samepairs of taxa between the two genes studied. First, distances betweenYersinia species are at least two times lower for atpD than for tuf(FIG. 10 c). Also, distances at the family level (betweenEnterobacteriaceae and Vibrionaceae) show that Enterobacteriaceae is atightlier knit family with atpD gene (Proteus genus excepted) than withtuf gene. Both genes well delineate taxa belonging to the same species.There is one exception with atpD: Klebsiella planticola and K.ornithinolithica belong to the same genus but fit with taxa belonging tothe same species (FIG. 10 a and c). These two species are also veryclose genotypically with tuf gene. This suggest that Klebsiellaplanticola and K. ornithinolithica could be two newborn species. tuf andatpD genes exhibit little distances between Escherichia fergusonii andE. coli-Shigella species. Unfortunately, comparison with 16S rDNA couldnot be achieved because the E. fergusonii 16S rDNA sequence is not yetaccessible in GenEMBL database. Therefore, the majority ofphenotypically close enterobacteria could be easily discriminatedgenotypically using tuf and atpD gene sequences.

In conclusion, tuf and atpD genes exhibit phylogenies consistent with16S rDNA genes phylogeny. For example, they reveal that the familyEnterobacteriaceae is monophyletic. Moreover, tuf and atpD distancesprovide a higher discriminating power than 16S rDNA distances. In fact,tuf and atpD genes discriminate well between different genospecies andare conserved between strains of the same genetic species in such a waythat primers and molecular probes for diagnostic purposes could bedesigned. Preliminary studies support these observations and diagnostictests based on tuf and atpD sequence data to identify enterobacteria arecurrently under development.

Example 44 Testing New Pairs of PCR Primers Selected from TwoSpecies-Specific Genomic DNA Fragments which are Objects of Our AssignedU.S. Pat. No. 6,001,564

Objective. The goal of these experiments is to demonstrate that it isrelatively easy for a person skilled in the art to find other PCR primerpairs from the species-specific fragments used as targets for detectionand identification of a variety of microorganisms. In fact, we wish toprove that the PCR primers previously tested by our group and which areobjects of the present patent application are not the only possible goodchoices for diagnostic purposes. For this example, we used diagnostictargets described in our assigned U.S. Pat. No. 6,001,564.

Experimental strategy. We have selected randomly two species-specificgenomic DNA fragments for this experiment. The first one is the 705-bpfragment specific to Staphylococcus epidermidis (SEQ ID NO: 36 from U.S.Pat. No. 6,001,564) while the second one is the 466-bp fragment specificto Moraxella catarrhalis (SEQ ID NO: 29 from U.S. Pat. No. 6,001,564).Subsequently, we have selected from these two fragments a number of PCRprimer pairs other than those previously tested. We have chosen 5 newprimer pairs from each of these two sequences which are well dispersedalong the DNA fragment (FIGS. 11 and 12). We have tested these primersfor their specificity and compared them with the original primerspreviously tested. For the specificity tests, we have tested allbacterial species closely related to the target species based onphylogenetic analysis with three conserved genes (rRNA genes, tuf andatpD). The rational for selecting a restricted number of bacterialspecies to evaluate the specificity of the new primer pairs is based onthe fact that the lack of specificity of a DNA-based assay isattributable to the detection of closely related species which are moresimilar at the nucleotide level. Based on the phylogenetic analysis, wehave selected (i) species from the closely related genus Staphylococcus,Enterococcus, Streptococcus and Listeria to test the specificity of theS. epidermidis-specific PCR assays and (ii) species from the closelyrelated genus Moraxella, Kingella and Neisseria to test the specificityof the M. catarrhalis-specific PCR assays.

Materials and Methods

Bacterial strains. All bacterial strains used for these experiments wereobtained from the American Type Culture Collection (ATCC, Rockville,Md.).

Genomic DNA isolation. Genomic DNA was purified from the ATCC referencestrains by using the G-nome DNA kit (Bio 101 Inc., Vista, Calif.).

Oligonucleotide design and synthesis. PCR primers were designed with thehelp of the Oligo™ primer analysis software Version 4.0 (NationalBiosciences Inc., Plymouth, Minn.) and synthesized using a model 391 DNAsynthesizer (Applied Biosystems, Foster City, Calif.).

PCR assays. All PCR assays were performed by using genomic DNA purifiedfrom reference strains obtained from the ATCC. One μl of purified DNApreparation (containing 0.01 to 1 ng of DNA per μl) was added directlyinto the PCR reaction mixture. The 20 μL PCR reactions contained finalconcentrations of 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100,2.5 mM MgCl₂, 0.4 μM of each primer, 200 μM of each of the four dNTPsand 0.5 unit of Taq DNA polymerase (Promega, Madison, Wis.) combinedwith the TaqStart™ antibody (Clontech Laboratories Inc., Palo Alto,Calif.). An internal control was integrated into all amplificationreactions to verify the efficiency of the amplification reaction as wellas to ensure that significant PCR inhibition was absent. Primersamplifying a region of 252 bp from a control plasmid added to eachamplification reaction were used to provide the internal control. PCRreactions were then subjected to thermal cycling (3 min at 95° C.followed by 30 cycles of 1 second at 95° C. for the denaturation stepand 30 seconds at 50 to 65° C. for the annealing-extension step) using aPTC-200 thermal cycler (MJ Research Inc., Watertown, Mass.). PCRamplification products were then analyzed by standard agarose gel (2%)electrophoresis. Amplification products were visualized in agarose gelscontaining 0.25 μg/mL of ethidium bromide under UV at 254 nm.

Results

Tables 21 and 22 show the results of specificity tests with the 5 newprimer pairs selected from SEQ ID NO: 29 (specific to M. catarrhalisfrom U.S. Pat. No. 6,001,564) and SEQ ID NO: 36 (specific to S.epidermidis from U.S. Pat. No. 6,001,564), respectively. In order toevaluate the performance of these new primers pairs, we compared them inparallel with the original primer pairs previously tested.

For M. catarrhalis, all of the 5 selected PCR primer pairs were specificfor the target species because none of the closely related species couldbe amplified (Table 21). In fact, the comparison with the originalprimer pair SEQ ID NO: 118+SEQ ID NO: 119 (from U.S. Pat. No. 6,001,564)revealed that all new pairs showed identical results in terms ofspecificity and sensitivity thereby suggesting their suitability fordiagnostic purposes.

For S. epidermidis, 4 of the 5 selected PCR primer pairs were specificfor the target species (Table 22). It should be noted that for 3 ofthese four primer pairs the annealing temperature had to be increasedfrom 55° C. to 60 or 65° C. to attain specificity for S. epidermidis.Again the comparison with the original primer pair SEQ ID NO: 145+SEQ IDNO: 146 (from U.S. Pat. No. 6,001,564) revealed that these four primerpairs were as good as the original pair. Increasing the annealingtemperature for the PCR amplification is well known by persons skilledin the art to be a very effective way to improve the specificity of aPCR assay (Persing et al., 1993, Diagnostic Molecular Microbiology:Principles and Applications, American Society for Microbiology,Washington, D.C.; Ehrlich and Greenberg, 1994, PCR-based Diagnostics inInfectious Disease, Blackwell Scientific Publications, Boston, Mass.).In fact, those skilled in the art are well aware of the fact that theannealing temperature is critical for the optimization of PCR assays.Only the primer pair VBsep3+VBsep4 amplified bacterial species otherthan S. epidermidis including the staphylococcal species S. capitis, S.cohnii, S. aureus, S. haemolyticus and S. hominis (Table 22). For thisnon-specific primer pair, increasing the annealing temperature from 55to 65° C. was not sufficient to attain the desired specificity. Onepossible explanation for the fact that it appears sligthly easier toselect species-specific primers for M. catarrhalis than for S.epidermidis is that M. catarrhalis is more isolated in phylogenetictrees than S. epidermidis. The large number of coagulase negativestaphylococcal species such as S. epidermidis is largely responsible forthis phylogenetic clustering.

Conclusion

These experiment clearly show that it is relatively easy for a personskilled in the art to select, from the species-specific DNA fragmentsselected as target for identification, PCR primer pairs suitable fordiagnostic purposes other than those previously tested. Theamplification conditions can be optimize by modifying critical variablessuch as the annealing temperature to attain the desired specificity andsensitivity. Consequently, we consider that it is legitimate to claimany possible primer sequences selected from the species-specificfragment and that it would be unfair to grant only the claims dealingwith the primer pairs previously tested. By extrapolation, these resultsstrongly suggest that it is also relatively easy for a person skilled inthe art to select, from the species-specific DNA fragments, DNA probessuitable for diagnostic purposes other than those previously tested.

Example 45 Testing Modified Versions of PCR Primers Derived from theSequence of Several Primers which are Objects of U.S. Pat. No.6,001,564.

Objective. The purpose of this project is to verify the efficiency ofamplification by modified PCR primers derived from primers previouslytested. The types of primer modifications to be tested include (i)variation of the sequence at one or more nucleotide positions and (ii)increasing or reducing the length of the primers. For this example, weused diagnostic targets described in U.S. Pat. No. 6,001,564.

Experimental Strategy:

Testing Primers with Nucleotide Changes

We have designed 13 new primers which are derived from the S.epidermidis-specific SEQ ID NO: 146 from U.S. Pat. No. 6,001,564 (Table23). These primers have been modified at one or more nucleotidepositions. As shown in Table 23, the nucleotide changes were introducedall along the primer sequence. Furthermore, instead of modifying theprimer at any nucleotide position, the nucleotide changes wereintroduced at the third position of each codon to better reflectpotential genetic variations in vivo. It should be noted that nonucleotide changes were introduced at the 3′ end of the oligonucleotideprimers because those skilled in the art are well aware of the fact thatmimatches at the 3′ end should be avoided (Persing et al., 1993,Diagnostic Molecular Microbiology: Principles and Applications, AmericanSociety for Microbiology, Washington, D.C.). All of these modifiedprimers were tested in PCR assays in combination with SEQ ID NO: 145from U.S. Pat. No. 6,001,564 and the efficiency of the amplification wascompared with the original primer pair SEQ ID NO: 145+SEQ ID NO: 146previously tested in U.S. Pat. No. 6,001,564.

Testing Shorter or Longer Versions of Primers

We have designed shorter and longer versions of the original S.epidermidis-specific PCR primer pair SEQ ID NO: 145+146 from U.S. Pat.No. 6,001,564 (Table 24) as well as shorter versions of the original P.aeruginosa-specific primer pair SEQ ID NO: 83+84 from U.S. Pat. No.6,001,564 (Table 25). As shown in Tables 24 and 25, both primers of eachpair were shortened or lengthen to the same length. Again, those skilledin the art know that the melting temperature of both primers from a pairshould be similar to avoid preferential binding at one primer bindingsite which is detrimental in PCR (Persing et al., 1993, DiagnosticMolecular Microbiology: Principles and Applications, American Societyfor Microbiology, Washington, D.C.; Ehrlich and Greenberg, 1994,PCR-based Diagnostics in Infectious Disease, Blackwell ScientificPublications, Boston, Mass.). All of these shorter or longer primerversions were tested in PCR assays and the efficiency of theamplification was compared with the original primer pair SEQ ID NOs 145and 146.

Materials and Methods

See the Materials and methods section of Example 44.

Results

Testing Primers with Nucleotide Changes

The results of the PCR assays with the 13 modified versions of SEQ IDNO: 146 from U.S. Pat. No. 6,001,564 are shown in Table 23. The 8modified primers having a single nucleotide variation showed anefficiency of amplification identical to the original primer pair basedon testing with 3 different dilutions of genomic DNA. The four primershaving two nucleotide variations and primer VBmut12 having 3 nucleotidechanges also showed PCR results identical to those obtained with theoriginal pair. Finally, primer VBmut13 with four nucleotide changesshowed a reduction in sensitivity by approximately one log as comparedwith the original primer pair. However, reducing the annealingtemperature from 55 to 50° C. gave an efficiency of amplification verysimilar to that observed with the original primer pair (Table 23). Infact, reducing the annealing temperature of PCR cycles represents aneffective way to reduce the stringency of hybridization for the primersand consequently allows the binding of probes with mismatches (Persinget al., 1993, Diagnostic Molecular Microbiology: Principles andApplications, American Society for Microbiology, Washington, D.C.).Subsequently, we have confirmed the specificity of the PCR assays witheach of these 13 modified versions of SEQ ID NO: 146 from U.S. Pat. No.6,001,564 by performing amplifications from all bacterial speciesclosely related to S. epidermidis which are listed in Table 22.

Testing Shorter or Longer Versions of Primers

For these experiments, two primer pairs were selected: i) SEQ ID NO:145+146 from U.S. Pat. No. 6,001,564 (specific to S. epidermidis) whichare AT rich and ii) SEQ ID NO: 83+84 (specific to P. aeruginosa) whichare GC rich. For the AT rich sequence, primers of 15 to 30 nucleotide inlength were designed (Table 24) while for the GC rich sequences, primersof 13 to 19 nucleotide in length were designed (Table 25).

Table 24 shows that, for an annealing temperature of 55° C., the 30-25-,20- and 17-nucleotide versions of SEQ ID NO: 145 and 146 from U.S. Pat.No. 6,001,564 all showed identical results as compared with the originalprimer pair except that the 17-nucleotide version amplified slightlyless efficiently the S. epidermidis DNA. Reducing the annealingtemperature from 55 to 45° C. for the 17-nucleotide version allowed toincrease the amplification efficiency to a level very similar to thatwith the original primer pair (SEQ ID NO: 145+146 from U.S. Pat. No.6,001,564). Regarding the 15-nucleotide version, there was amplificationof S. epidermidis DNA only when the annealing temperature was reduced to45° C. Under those PCR conditions the assay remained S.epidermidis-specific but the amplification signal with S. epidermidisDNA was sligthly lower as compared with the original primer pair.Subsequently, we have further confirmed the specificity of the shorteror longer versions by amplifying DNA from all bacterial species closelyrelated to S. epidermidis which are listed in Table 22.

Table 25 shows that, for an annealing temperature of 55° C., all shorterversions of SEQ ID NO: 83 and 84 from U.S. Pat. No. 6,001,564 showedidentical PCR results as compared with the original primer pair. Asexpected, these results show that it is simpler to reduce the length ofGC rich as compared with AT rich. This is attributable to the fact thatGC binding is more stable than AT binding.

Conclusion

Testing Primers with Nucleotide Changes

The above experiments clearly show that PCR primers may be modified atone or more nucleotide positions without affecting the specificity andthe sensitivity of the PCR assay. These results strongly suggest that agiven oligonucleotide can detect variant genomic sequences from thetarget species. In fact, the nucleotide changes in the selected primerswere purposely introduced at the third position of each codon to mimicnucleotide variation in genomic DNA. Thus we conclude that it isjustified to claim “a variant thereof” for i) the SEQ IDs of thefragments and oligonucleotides which are object of the present patentapplication and ii) genomic variants of the target species.

Testing Shorter or Longer Versions of Primers

The above experiments clearly show that PCR primers may be shorter orlonger without affecting the specificity and the sensitivity of the PCRassay. We have showed that oligonucleotides ranging in sizes from 13 to30 nucleotides may be as specific and sensitive as the original primerpair from which they were derived. Consequently, these results suggestthat it is not exaggerated to claim sequences having at least 12nucleotide in length.

This invention has been described herein above, and it is readilyapparent that modifications can be made thereto without departing fromthe spirit of this invention. These modifications are under the scope ofthis invention, as defined in the appended claims.

TABLE 1 Distribution (%) of nosocomial pathogens for various humaninfections in USA (1990-1992)¹. Pathogen UTI² SSI³ BSI⁴ Pneumonia CSF⁵Escherichia coli 27 9 5 4 2 Staphylococcus aureus 2 21 17 21 2Staphylococcus epidermidis 2 6 20 0 1 Enterococcus faecalis 16 12 9 2 0Enterococcus faecium 1 1 0 0 0 Pseudomonas aeruginosa 12 9 3 18 0Klebsiella pneumoniae 7 3 4 9 0 Proteus mirabilis 5 3 1 2 0Streptococcus pneumoniae 0 0 3 1 18 Group B Streptococci 1 1 2 1 6 Otherstreptococci 3 5 2 1 3 Haemophilus influenzae 0 0 0 6 45 Neisseriameningitidis 0 0 0 0 14 Listeria monocytogenes 0 0 0 0 3 Otherenterococci 1 1 0 0 0 Other staphylococci 2 8 13 2 0 Candida albicans 93 5 5 0 Other Candida 2 1 3 1 0 Enterobacter sp. 5 7 4 12 2Acinetobacter sp. 1 1 2 4 2 Citrobacter sp. 2 1 1 1 0 Serratiamarcescens 1 1 1 3 1 Other Klebsiella 1 1 1 2 1 Others 0 6 4 5 0 ¹Datarecorded by the National Nosocomial Infections Surveillance (NNIS) from80 hospitals (Emori and Gaynes, 1993, Clin. Microbiol. Rev., 6:428-442).²Urinary tract infection. ³Surgical site infection. ⁴Bloodstreaminfection. ⁵Cerebrospinal fluid.

TABLE 2 Distribution (%) of bloodstream infection pathogens in Quebec(1995), Canada (1992), UK (1969-1988) and USA (1990-1992). UK³ USA⁴Community- Hospital- Hospital- Organism Quebec¹ Canada² acquiredacquired acquired E. coli 15.6 53.8 24.8 20.3 5.0 S. epidermitis and25.8 — 0.5 7.2 31.0 other CoNS⁵ S. aureus 9.6 — 9.7 19.4 16.0 S.pneumoniae 6.3 — 22.5 2.2 — E. faecalis 3.0 — 1.0 4.2 — E. faecium 2.6 —0.2 0.5 — Enterococcus sp. — — — — 9.0 H. influenzae 1.5 — 3.4 0.4 — P.aeruginosa 1.5 8.2 1.0 8.2 3.0 K. pneumoniae 3.0 11.2 3.0 9.2 4.0 P.mirabilis — 3.9 2.8 5.3 1.0 S. pyogenes — — 1.9 0.9 — Enterobacter sp.4.1 5.5 0.5 2.3 4.0 Candida sp. 8.5 — — 1.0 8.0 Others 18.5 17.4 28.718.9 19.0 ¹Data obtained for 270 isolates collected at the CentreHospitalier de l'Universite Laval (CHUL) during a 5 month period (May toOctober 1995). ²Data from 10 hospitals throughout Canada representing941 gram-negative isolates. (Chamberland et al., 1992, Clin. Infect.Dis., 15:615-628). ³Data from a 20-year study (1969-1988) for nearly4000 isolates. (Eykyn et al., 41990, J. Antimicrob. Chemother., Suppl.C, 25:41-58). ⁴Data recorded by the National Nosocomial InfectionsSurveillance (NNIS) from 80 hospitals (Emori and Gaynes, 1993, Clin.Microbiol. Rev., 6:428-442). ⁵Coagulase-negative staphylococci.

TABLE 3 Distribution of positive and negative clinical specimens testedat the microbiology laboratory of the CHUL (February 1994-January 1995).Clinical No. of % of % of specimens samples positive negative and/orsites tested (%) specimens specimens Urine 17,981 (54.5) 19.4 80.6 Bloodculture/marrow 10,010 (30.4) 6.9 93.1 Sputum  1,266 (3.8) 68.4 31.6Superficial pus  1,136 (3.5) 72.3 27.7 Cerebrospinal fluid   553 (1.7)1.0 99.0 Synovial fluid   523 (1.6) 2.7 97.3 Respiratory tract   502(1.5) 56.6 43.4 Deep pus   473 (1.4) 56.8 43.2 Ears   289 (0.9) 47.152.9 Pleural and pericardial fluid   132 (0.4) 1.0 99.0 Peritoneal fluid  101(0.3) 28.6 71.4 Total: 32,966 (100.0) 20.0 80.0

TABLE 4 Example of microbial species for which tuf and/or atpD and/orrecA nucleic acids and/or sequences are used in the present invention.Bacterial species Abiotrophia adiacens Abiotrophia defectivaAchromobacter xylosoxidans subsp. denitrificans Acetobacterium woodiAcetobacter aceti Acetobacter altoacetigenes Acetobacter polyoxogenesAcholeplasma laidlawii Acidothermus cellulolyticus Acidiphilum facilisAcinetobacter baumannii Acinetobacter calcoaceticus Acinetobacterlwoffii Actinomyces meyeri Aerococcus viridans Aeromonas hydrophilaAeromonas salmonicida Agrobacterium radiobacter Agrobacteriumtumefaciens Alcaligenes faecalis subsp. faecalis Allochromatium vinosumAnabaena variabilis Anacystis nidulans Anaerorhabdus furcosus Aquifexaeolicus Aquifex pyrophilus Arcanobacterium haemolyticum Archaeoglobusfulgidus Azotobacter vinelandii Bacillus anthracia Bacillus cereusBacillus firmus Bacillus halodurans Bacillus megaterium Bacillusmycoides Bacillus pseudomycoides Bacillus stearothermophilus Bacillussubtilis Bacillus thuringiensis Bacillus weihenstephanensis Bacteroidesdistasonis Bacteroides fragilis Bacteroides forsythus Bacteroides ovatusBacteroides vulgatus Bartonella henselae Bifidobacterium adolescentisBifidobacterium breve Bifidobacterium dentium Bifidobacterium longumBlastochloris viridis Borrelia burgdorferi Bordetella pertussisBordetella bronchiseptica Brucella abortus Brevibacterium linensBrevibacterium flavum Brevundimonas diminuta Buchnera aphidicolaBudvicia aquatica Burkholderia cepacia Burkholderia mallei Burkholderiapseudomallei Buttiauxella agrestis Butyrivibrio fibrisolvensCampylobacter coli Campylobacter curvus Campylobacter fetus subsp. fetusCampylobacter fetus subsp. venerealis Campylobacter gracilisCampylobacter jejuni Campylobacter jejuni subsp. doylei Campylobacterjejuni subsp. jejuni Campylobacter lari Campylobacter rectusCampylobacter sputorum subsp. sputorum Campylobacter upsaliensis Cedeceadavisae Cedecea lapagei Cedecea neteri Chlamydia pneumoniae Chlamydiapsittaci Chlamydia trachomatis Chlorobium vibrioforme Chloroflexusaurantiacus Chryseobacterium meningosepticum Citrobacter amalonaticusCitrobacter braakii Citrobacter farmeri Citrobacter freundii Citrobacterkoseri Citrobacter sedlakii Citrobacter werkmanii Citrobacter youngaeClostridium acetobutylicum Clostridium beuerinckii Clostridiumbifermentans Clostridium botulinum Clostridium difficile Clostridiuminnocuum Clostridium histolyticum Clostridium novyi Clostridium septicumClostridium perfringens Clostridium ramosum Clostridium tertiumClostridium tetani Comamonas acidovorans Corynebacterium accolensCorynebacterium bovis Corynebacterium cervicis Corynebacteriumdiphtheriae Corynebacterium flavescens Corynebacterium genitaliumCorynebacterium glutamicum Corynebacterium jeikeium Corynebacteriumkutscheri Corynebacterium minutissimum Corynebacterium mycetoidesCorynebacterium pseudodiphtheriticum Corynebacterium pseudo genitaliumCorynebacterium pseudotuberculosis Corynebacterium renaleCorynebacterium striatum Corynebacterium ulcerans Corynebacteriumurealyticum Corynebacterium xerosis Coxiella burnetii Cytophaga lyticaDeinococcus radiodurans Deinonema sp. Edwardsiella hoshinae Edwardsiellatarda Ehrlichia canis Ehrlichia risticii Eikenella corrodensEnterobacter aerogenes Enterobacter agglomerans Enterobacter amnigenusEnterobacter asburiae Enterobacter cancerogenus Enterobacter cloacaeEnterobacter gergoviae Enterobacter hormaechei Enterobacter sakazakiiEnterococcus avium Enterococcus casseliflavus Enterococcus cecorumEnterococcus columbae Enterococcus dispar Enterococcus duransEnterococcus faecalis Enterococcus faecium Enterococcus flavescensEnterococcus gallinarum Enterococcus hirae Enterococcus malodoratusEnterococcus mundtii Enterococcus pseudoavium Enterococcus raffinosusEnterococcus saccharolyticus Enterococcus solitarius Enterococcussulfureus Clostridium sordellii Erwinia amylovora Erwinia carotovoraEscherichia coli Escherichia fergusonii Escherichia hermanniiEscherichia vulneris Eubacterium lentum Eubacterium nodatum Ewingellaamericana Francisella tularensis Frankia alni Fervidobacteriumislandicum Fibrobacter succinogenes Flavobacterium ferrigeneumFlexistipes sinusarabici Fusobacterium gonidiaformans Fusobacteriumnecrophorum subsp. necrophorum Fusobacterium nucleatum subsp.polymorphum Gardnerella vaginalis Gemella haemolysans Gemellamorbillorum Globicatella sanguis Gloeobacter violaceus Gloeothece sp.Gluconobacter oxydans Haemophilus actinomycetemcomitans Haemophilusaphrophilus Haemophilus ducreyi Haemophilus haemolyticus Haemophilusinfluenzae Haemophilus parahaemolyticus Haemophilus parainfluenzaeHaemophilus paraphrophilus Haemophilus segnis Hafnia alvei Halobacteriummarismortui Halobacterium salinarum Haloferax volcanii Helicobacterpylori Herpetoshiphon aurantiacus Kingella kingae Klebsiellaornithinolytica Klebsiella oxytoca Klebsiella planticola Klebsiellapneumoniae subsp. ozaenae Klebsiella pneumoniae subsp. pneumoniaeKlebsiella pneumoniae subsp. rhinoscleromatis Klebsiella terrigenaKluyvera ascorbata Kluyvera cryocrescens Kluyvera georgiana Kocuriakristinae Lactobacillus acidophilus Lactobacillus garvieae Lactobacillusparacasei Lactobacillus casei subsp. casei Lactococcus garvieaeLactococcus lactis Lactococcus lactis subsp. lactis Legionella micdadeiLegionella pneumophila subsp. pneumophila Leminorella grimontiiLeminorella richardii Leptospira biflexa Leptospira interrogansLeuconostoc mesenteroides subsp. dextranicum Listeria innocua Listeriaivanovii Listeria monocytogenes Listeria seeligeri Macrococcuscaseolyticus Magnetospirillum magnetotacticum Megamonas hypermegaleMethanobacterium thermoautotrophicum Methanococcus jannaschiiMethanococcus vannielii Methanosarcina barkeri Methanosarcina jannaschiiMethylobacillus flagellatum Methylomonas clara Micrococcus luteusMicrococcus lylae Mitsuokella multacidus Mobiluncus curtisii subsp.holmesii Moellerella thermoacetica Moellerella wisconsensis Moorellathermoacetica Moraxella catarrhalis Moraxella osloensis Morganellamorganii subsp. morganii Mycobacterium avium Mycobacterium bovisMycobacterium gordonae Mycobacterium kansasii Mycobacterium lepraeMycobacterium terrae Mycobacterium tuberculosis Mycoplasma capricolumMycoplasma gallisepticum Mycoplasma genitalium Mycoplasma hominisMycoplasma pirum Mycoplasma mycoides Mycoplasma pneumoniae Mycoplasmapulmonis Mycoplasma salivarium Myxococcus xanthus Neisseria animalisNeisseria canis Neisseria cinerea Neisseria cuniculi Neisseria elongatasubsp. elongata Neisseria elongata subsp. intermedia Neisseria flavaNeisseria flavescens Neisseria gonorrhoeae Neisseria lactamica Leclerciaadecarboxylata Neisseria meningitidis Neisseria mucosa Neisseriaperflava Neisseria pharyngis var.flava Neisseria polysaccharea Neisseriasicca Neisseria subflava Neisseria weaveri Obesumbacterium proteusOchrobactrum anthropi Pantoea agglomerans Pantoea dispersa Paracoccusdenitrificans Pasteurella multocida Pectinatus frisingensis Peptococcusniger Peptostreptococcus anaerobius Peptostreptococcus asaccharolyticusPeptostreptococcus prevotii Phormidium ectocarpi Pirellula marinaPlanobispora rosea Plesiomonas shigelloides Plectonema boryanumPorphyromonas asaccharolytica Porphyromonas gingivalis Pragia fontiumPrevotella buccalis Prevotella melaninogenica Prevotella oralisPrevotella ruminocola Prochlorothrix hollandica Propionibacterium acnesPropionigenium modestum Proteus mirabilis Proteus penneri Proteusvulgaris Providencia alcalifaciens Providencia rettgeri Providenciarustigianii Providencia stuartii Pseudomonas aeruginosa Pseudomonasfluorescens Pseudomonas putida Pseudomonas stutzeri Psychrobacterphenylpyruvicum Pyrococcus abyssi Rahnella aquatilis Rickettsiaprowazekii Rhizobium leguminosarum Rhizobium phaseoli Rhodobactercapsulatus Rhodobacter sphaeroides Rhodopseudomonas palustrisRhodospirillum rubrum Ruminococcus albus Ruminococcus bromii Salmonellabongori Salmonella choleraesuis subsp. arizonae Salmonella choleraesuissubsp choleraesuis Salmonella choleraesuis subsp. diarizonae Salmonellacholeraesuis subsp. houtenae Salmonella choleraesuis subsp. indicaSalmonella choleraesuis subsp. salamae Serpulina hyodysenteriae Serratiaficaria Serratia fonticola Serratia grimesii Serratia liquefaciensSerratia marcescens Serratia odorifera Serratia plymuthica Serratiarubidaea Shewanella putrefaciens Shigella boydii Shigella dysenteriaeShigella flexneri Shigella sonnei Sinorhizobium meliloti Spirochaetaaurantia Staphylococcus aureus Staphylococcus aureus subsp. aureusStaphylococcus auricularis Staphylococcus capitis subsp. capitisStaphylococcus cohnii subsp. cohnii Staphylococcus epidermidisStaphylococcus haemolyticus Staphylococcus hominis Staphylococcushominis subsp. hominis Staphylococcus lugdunensis Staphylococcussaprophyticus Staphylococcus sciuri subsp. sciuri Staphylococcussimulans Staphylococcus warneri Stigmatella aurantiaca Stenotrophomonasmaltophilia Streptococcus acidominimus Streptococcus agalactiaeStreptococcus anginosus Streptococcus bovis Streptococcus cricetusStreptococcus cristatus Streptococcus downei Streptococcus dysgalactiaeStreptococcus equi subsp. equi Streptococcus ferus Streptococcusgordonii Streptococcus macacae Streptococcus mitis Streptococcus mutansStreptococcus oralis Streptococcus parasanguinis Streptococcuspneumoniae Streptococcus pyogenes Streptococcus ratti Streptococcussalivarius Streptococcus salivarius subsp. thermophilus Streptococcussanguinis Streptococcus sobrinus Streptococcus suis Streptococcus uberisStreptococcus vestibularis Streptomyces anbofaciens Streptomycesaureofaciens Streptomyces cinnamoneus Streptomyces coelicolorStreptomyces collinus Streptomyces lividans Streptomyces netropsisStreptomyces ramocissimus Streptomyces rimosus Streptomyces venezuelaeSuccinivibrio dextrinosolvens Synechococcus sp. Synechocystis sp.Tatumella ptyseos Taxeobacter occealus Tetragenococcus halophilusThermoplasma acidophilum Thermotoga maritima Thermus aquaticus Thermusthermophilus Thiobacillus ferrooxidans Thiomonas cuprina Trabulsiellaguamensis Treponema pallidum Ureaplasma urealyticum Veillonella parvulaVibrio alginolyticus Vibrio anguillarum Vibrio cholerae Vibrio mimicusWolinella succinogenes Xanthomonas cirri Xanthomonas oryzae Xenorhabdusbovieni Xenorhabdus nematophilus Yersinia bercovieri Yersiniaenterocolitica Yersinia frederiksensii Yersinia intermedia Yersiniapestis Yersinia pseudotuberculosis Yersinia rohdei Yokenellaregensburgei Zoogloea ramigera Fungal species Absidia corymbiferaAbsidia glauca Alternaria alternata Arxula adeninivorans Aspergillusflavus Aspergillus fumigatus Aspergillus nidulans Aspergillus nigerAspergillus oryzae Aspergillus terreus Aspergillus versicolorAureobasidium pullulans Basidiobolus ranarum Bipolaris hawaiiensisBilophila wadsworthia Blastoschizomyces capitates Blastomycesdermatitidis Candida albicans Candida catenulata Candida dubliniensisCandida famata Candida glabrata Candida guilliermondii Candidahaemulonii Candida inconspicua Candida kefyr Candida krusei Candidalambica Candida lusitaniae Candida norvegica Candida norvegensis Candidaparapsilosis Candida rugosa Candida sphaerica Candida tropicalis Candidautilis Candida viswanathii Candida zeylanoides Cladophialophoracarrionii Coccidioides immitis Coprinus cinereus Cryptococcus albidusCryptococcus humicolus Cryptococcus laurentii Cryptococcus neoformansCunninghamella bertholletiae Curvularia lunata Emericella nidulansEmmonsia parva Eremothecium gossypii Exophiala dermatitidis Exophialajeanselmei Exophiala moniliae Exserohilum rostratum Eremotheciumgossypii Fonsecaea pedrosoi Fusarium moniliforme Fusarium oxysporumFusarium solani Geotrichum sp. Histoplasma capsulatum Hortaea werneckiiIssatchenkia orientalis Kudrjanzev Kluyveromyces lactic Malasseziafurfur Malassezia pachydermatis Malbranchea filamentosa Metschnikowiapulcherrima Microsporum audouinii Microsporum canis Mucor circinelloidesNeurospora crassa Paecilomyces lilacinus Paracoccidioides brasiliensisPenicillium marneffei Phialaphora verrucose Pichia anomala Piedraiahortai Podospora anserina Podospora curvicolla Puccinia graminisPseudallescheria boydii Reclinomonas americana Rhizomucor racemosusRhizopus oryzae Rhodotorula minuta Rhodotorula mucilaginosaSaccharomyces cerevisiae Saksenaea vasiformis Schizosaccharomyces pombeScopulariopsis koningii Sordaria macrospora Sporobolomyces salmonicolorSporothrix schenckii Stephanoascus ciferrii Syncephalastrum racemosumTrichoderma reesei Trichophyton mentagrophytes Trichophyton rubrumTrichophyton tonsurans Trichosporon cutaneum Ustilago maydis Wangielladermatitidis Yarrowia lipolytica Parasitical species Babesia bigeminaBabesia bovis Babesia microti Blastocystis hominis Crithidia fasciculataCryptosporidium parvum Entamoeba histolytica Giardia lambliaKentrophoros sp. Leishmania aethiopica Leishmania amazonensis Leishmaniabraziliensis Leishmania donovani Leishmania infantum Leishmaniaenriettii Leishmania gerbilli Leishmania guyanensis Leishmania hertigiLeishmania major Leishmania mexicana Leishmania panamensis Leishmaniatarentolae Leishmania tropica Neospora caninum Onchocerca volvulusPlasmodium berghei Plasmodium falciparum Plasmodium knowlesi Porphyrapurpurea Toxoplasma gondii Treponema pallidum Trichomonas tenaxTrichomonas vaginalis Trypanosoma brucei Trypanosoma brucei subsp.brucei Trypanosoma congolense Trypanosoma cruzi

TABLE 5 Antimicrobial agents resistance genes selected for diagnosticpurposes Gene Antimicrobial ACCESSION ID NO. agent Bacteria¹ NO. SEQ IDNO. aac(3)-Ib² Aminoglycosides Enterobacteriaceae L06157 Pseudomonadsaac(3)-IIb² Aminoglycosides Enterobacteriaceae, M97172 Pseudomonadsaac(3)-IVa² Aminoglycosides Enterobacteriaceae X01385 aac(3)-VIa²Aminoglycosides Enterobacteriaceae, M88012 Pseudomonads aac(2′)-1a²Aminoglycosides Enterobacteriaceae, X04555 Pseudomonads aac(6′)-aph(2″)²Aminoglycosides Enterococcus sp., 83-86³ Staphylococcus sp. aac(6′)-Ia,²Aminoglycosides Enterobacteriaceae, M18967 Pseudomonads aac(6′)-Ic²Aminoglycosides Enterobacteriaceae, M94066 Pseudomonads aac(6′)-IIa²Aminoglycosides Pseudomonads 112⁴ aadB AminoglycosidesEnterobacteriaceae 53-54³ [ant(2″)-Ia²] aacC1 AminoglycosidesPseudomonads 55-56³ [aac(3)-Ia²] aacC2 Aminoglycosides Pseudomonads57-58³ [aac(3)-IIa²] aacC3 Aminoglycosides Pseudomonads 59-60³[aac(3)-III²] aacA4 Aminoglycosides Pseudomonads 65-66³ [aac(6′)-Ib²]ant(3″)-Ia² Aminoglycosides Enterobacteriaceae, X02340 Enterococcus sp.,M10241 Staphylococcus sp. ant(4′)-Ia² Aminoglycosides Staphylococcus sp.V01282 aph(3′)-Ia² Aminoglycosides Enterobacteriaceae, J01839Pseudomonads aph(3′)-IIa² Aminoglycosides Enterobacteriaceae, V00618Pseudomonads aph(3′)-IIIa² Aminoglycosides Enterococcus sp., V01547Staphylococcus sp. aph(3′)-VIa² Aminoglycosides Enterobacteriaceae,X07753 Pseudomonads rpsL² Streptomycin M. tuberculosis, X80120 M. aviumcomplex U14749 X70995 L08011 ^(bla)OXA^(5,6) β-lactamsEnterobacteriaceae, Y10693 110⁴ Pseudomonads AJ238349 AJ009819 X06046X03037 X07260 U13880 X75562 AF034958 J03427 Z22590 U59183 L38523 U63835AF043100 AF060206 U85514 AF043381 AF024602 AF064820 ^(bla)ROB⁵ β-lactamsHaemophilus sp. 45-48³ ^(bla)SHV^(5,6) β-lactams Enterobacteriacea,AF124984 41-44³ Pseudomonas AF148850 aeruginosa M59181 X98099 M33655AF148851 X53433 L47119 AF074954 X53817 AF096930 X55640 Y11069 U20270U92041 S82452 X98101 X98105 AF164577 AJ011428 AF116855 AB023477 AF293345AF227204 AF208796 AF132290 ^(bla)TEM^(5,6) β-lactams Enterobacteriaceae,AF012911 37-40³ Neisseria sp., U48775 Haemophilus sp. AF093512 AF052748X64523 Y13612 X57972 AF157413 U31280 U36911 U48775 V00613 X97254AJ012256 X04515 AF126482 U09188 M88143 Y14574 AF188200 AJ251946 Y17581Y17582 Y17583 M88143 U37195 Y17584 X64523 U95363 Y10279 Y10280 Y10281AF027199 AF104441 AF104442 AF062386 X57972 AF047171 AF188199 AF157553AF190694 AF190695 AF190693 AF190692 ^(bla)SHV^(5,6) β-lactamsEnterobacteriacea, AF124984 41-44³ Pseudomonas AF148850 aeruginosaM59181 X98099 M33655 AF148851 X53433 L47119 AF074954 X53817 AF096930X55640 Y11069 U20270 U92041 S82452 X98101 X98105 AF164577 AJ011428AF116855 AB023477 AF293345 AF227204 AF208796 AF132290 ^(bla)TEM^(5,6)β-lactams Enterobacteriaceae, AF012911 37-40³ Neisseria sp., U48775Haemophilus sp. AF093512 AF052748 X64523 Y13612 X57972 AF157413 U31280U36911 U48775 V00613 X97254 AJ012256 X04515 AF126482 U09188 M88143Y14574 AF188200 AJ251946 Y17581 Y17582 Y17583 M88143 U37195 Y17584X64523 U95363 Y10279 Y10280 Y10281 AF027199 AF104441 AF104442 AF062386X57972 AF047171 AF188199 AF157553 AF190694 AF190695 AF190693 AF190692^(bla)CARB⁵ β-lactams Pseudomonas sp., J05162 Enterobacteriaceae S46063M69058 U14749 D86225 D13210 Z18955 AF071555 AF153200 AF030945^(bla)CTX-M-1⁵ β-lactams Enterobacteriaceae X92506 ^(bla)CTX-M-2⁵β-lactams Enterobacteriaceae X92507 ^(bla)CMY-2 β-lactamsEnterobacteriaceae X91840 AJ007826 AJ011293 AJ011291 Y17716 Y16783Y16781 Y15130 U77414 S83226 Y15412 X78117 ^(bla)Imp⁵ β-lactamsEnterobacteriaceae, AJ223604 Pseudomonas S71932 aeruginosa D50438 D29636X98393 AB010417 D78375 ^(bla)PER-1⁵ β-lactams Enterobacteriaceae, Z21957Pseudomodanaceae ^(bla)PER-2⁷ β-lactams Enterobacteriaceae X93314 blaZ¹²β-lactams Enterococcus sp., 111⁴ Staphylococcus sp. mecA¹² β-lactamsStaphylococcus sp. 97-98³ pbp1a¹³ β-lactams Streptococcus M905271004-1018, pneumoniae X67872 1648, AB006868 2056-2064, AB0068742273-2276 X67873 AB006878 AB006875 AB006877 AB006879 AF046237 AF046235AF026431 AF046232 AF046233 AF046236 X67871 Z49095 AF046234 AB006873X67866 X67868 AB006870 AB006869 AB006872 X67870 AB006871 X67867 X67869AB006876 AF046230 AF046238 Z49094 pbp2b¹³ β-lactams Streptococcus X160221019-1033 pneumoniae M25516 M25518 M25515 U20071 U20084 U20082 U20067U20079 Z22185 U20072 pbp2b¹³ β-lactams Streptococcus U20083 pneumoniaeU20081 M25522 U20075 U20070 U20077 U20068 Z22184 U20069 U20078 M25521M25525 M25519 Z21981 M25523 M25526 U20076 U20074 M25520 M25517 M25524Z22230 U20073 U20080 pbp2x¹³ β-lactams Streptococcus X16367 1034-1048pneumoniae X65135 AB011204 AB011209 AB011199 AB011200 AB011201 AB011202AB011198 AB011208 AB011205 AB015852 AB011210 AB015849 AB015850 AB015851AB015847 AB015846 AB011207 AB015848 Z49096 int β-lactams,Enterobacteriaceae, 99-102³ trimethoprim sul aminoglycosides,Pseudomonads 103-106³ antiseptic, chloramphenicol ermA¹⁴ Macrolides,Staphylococcus sp. 113⁴ lincosamides, streptogramin B ermB¹⁴ Macrolides,Enterobacteriaceae, 114⁴ lincosamides, Staphylococcus sp. streptograminB Enterococcus sp. Streptococcus sp. ermC¹⁴ Macrolides,Enterobacteriaceae, 115⁴ lincosamides, Staphylococcus sp. streptograminB ereA¹² Macrolides Enterobacteriaceae, M11277 Staphylococcus sp. E01199AF099140 ereB¹² Macrolides Enterobacteriaceae A15097 Staphylococcus sp.X03988 msrA¹² Macrolides Staphylococcus sp. 77-80³ mefA, mefE⁸Macrolides Streptococcus sp. U70055 U83667 mphA⁸ MacrolidesEnterobacteriaceae, D16251 Staphylococcus sp. U34344 U36578 linA/linA'⁹Lincosamides Staphylococcus sp. J03947 M14039 A15070 E01245 linB¹⁰Lincosamides Enterococcus AF110130 faecium AJ238249 vga¹⁵ StreptrograminStaphylococcus sp. M90056 89-90³ U82085 vgb¹⁵ StreptrograminStaphylococcus sp. M36022 M20219 AF015628 vat¹⁵ StreptrograminStaphylococcus sp. L07778 87-88³ vatB¹⁵ Streptrogramin Staphylococcussp. U19459 L38809 satA¹⁵ Streptrogramin Enterococcus faecium L1203381-82³ mupA¹² Mupirocin Staphylococcus X75439 aureus X59478 X59477gyrA¹⁶ Quinolones Gram-positive and X95718 1255, gram-negative X067441607-1608, bacteria X57174 1764-1776, X16817 2013-2014, X71437 2277-2280AF065152 AF060881 D32252 parC/grlA¹⁶ Quinolones Gram-positive andAB005036 1777-1785 gram-negative AF056287 bacteria X95717 AF129764AB017811 AF065152 parE/grlB¹⁶ Quinolones Gram-positive X95717 bacteriaAF065153 AF058920 norA¹⁶ Quinolones Staphylococcus sp. D90119 M80252M97169 mexR(nalB)¹⁶ Quinolones Pseudomonas U23763 aeruginosa nfxB¹⁶Quinolones Pseudomonas X65646 aeruginosa cat¹² ChloramphenicolGram-positive and M55620 gram-negative X15100 bacteria A24651 M28717A00568 A00569 X74948 Y00723 A24362 A00569 M93113 M62822 M58516 V01277X02166 M77169 X53796 J01841 X07848 ppflo-like Chloramphenicol AF071555embB¹⁷ Ethambutol Mycobacterium U68480 tuberculosis pncA¹⁷ PyrazinamideMycobacterium U59967 tuberculosis rpoB¹⁷ Rifampin Mycobacterium AF055891tuberculosis AF055892 S71246 L27989 AF055893 inhA¹⁷ IsoniazidMycobacterium AF106077 tuberculosis U02492 vanA¹² VancomycinEnterococcus sp. 67-70³ 1049-1057 vanB¹² Vancomycin Enterococcus sp.116⁴ vanC1¹² Vancomycin Enterococcus 117⁴ gallinarum 1058-1059 vanC2¹²Vancomycin Enterococcus U94521 1060-1063 casseliflavus U94522 U94523U94524 U94525 L29638 vanC3¹² Vancomycin Enterococcus L29639 1064-1066flavescens U72706 vanD¹⁸ Vancomycin Enterococcus AF130997 faecium vanE¹²Vancomycin Enterococcus AF136925 faecium tetB¹⁹ TetracyclineGram-negative J01830 bacteria AF162223 AP000342 S83213 U81141 V00611tetM¹⁹ Tetracycline Gram-negative and X52632 Gram-positive AF116348bacteria U50983 X92947 M211136 U08812 X04388 sul II²⁰ SulfonamidesGram-negative M36657 bacteria AF017389 AF017391 dhfrla²⁰ TrimethoprimGram-negative AJ238350 bacteria X17477 K00052 U09476 X00926 dhfrlb²⁰Trimethoprim Gram-negative Z50805 bacteria Z50804 dhfrV²⁰ TrimethoprimGram-negative X12868 bacteria dhfrVI²⁰ Trimethoprim Gram-negative Z86002bacteria dhfrVII²⁰ Trimethoprim Gram-negative U31119 bacteria AF139109X58425 dhfrVIII²⁰ Trimethoprim Gram-negative U10186 bacteria U09273dhfrIX²⁰ Trimethoprim Gram-negative X57730 bacteria dhfrXII²⁰Trimethoprim Gram-negative Z21672 bacteria AF175203 AF180731 M84522dhfrXIII²⁰ Trimethoprim Gram-negative Z50802 bacteria dhfrXV²⁰Trimethoprim Gram-negative Z83331 bacteria dhfrXVII²⁰ TrimethoprimGram-negative AF170088 bacteria AF180469 AF169041 dfrA²⁰ TrimethoprimStaphylococcus sp. AF045472 U40259 AF051916 X13290 Y07536 Z16422 Z48233¹Bacteria having high incidence for the specified antibiotic resistancegene. The presence of the antibiotic resistance genes in other bacteriais not excluded. ²Shaw, K. J., P. N. Rather, R. S. Hare, and G. H.Miller. 1993. Molecular genetics of aminoglycoside resistance genes andfamilial relationships of the aminoglycoside-modifying enzymes.Microbiol. Rev. 57:138-163. ³Antibiotic resistance genes from ourassigned U.S. Pat. No. 6,001,564 for which we have selected PCR primerpairs. ⁴These SEQ ID NOs. refer to a previous patent (publicationW098/20157). ⁵Bush, K., G. A. Jacoby and A. Medeiros. 1995. A functionalclassification scheme for β-lactamase and its correlation with molecularstructure. Antimicrob. Agents. Chemother. 39:1211-1233. ⁶Nucleotidemutations in bla_(SHV), bla_(TEM), and bla_(OXA), are associated withextended-spectrum β-lactamase or inhibitor-resistant β-lactamase.⁷Bauerfeind, A., Y. Chong, and K. Lee. 1998. Plasmid-encoded AmpCbeta-lactamases: how far have we gone 10 ears after discovery? YonseiMed. J. 39:520-525. ⁸Sutcliffe, J., T. Grebe, A. Tait-Kamradt, and L.Wondrack. 1996. Detection of erythromycin-resistant determinants by PCR.Antimicrob. Agent Chemother. 40:2562-2566. ⁹Leclerc, R., A.,Brisson-Noël, J. Duval, and P. Courvalin. 1991. Phenotypic expressionand genetic heterogeneity of lincosamide inactivation in Staphylococcussp. Antimicrob. Agents. Chemother. 31:1887-1891. ¹⁰Bozdogan, B., L.Berrezouga, M.-S. Kuo, D. A. Yurek, K. A. Farley, B. J. Stockman, and R.Leclercq. 1999. A new gene, linB, conferring resistance to lincosamidesby nucleotidylation in Enterococcus faecium HM1025. Antimicrob. Agents.Chemother. 43:925-929. ¹¹Cockerill III, F.R. 1999. Genetic methods forassessing antimicrobial resistance. Antimicrob. Agents. Chemother.43:199-212. ¹²Tenover, F. C., T. Popovic, and O Olsvik. 1996. Geneticmethods for detecting antibacterial resistance genes. pp. 1368-1378. InMurray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover, R. H. Yolken(eds). Manual of clinical microbiology. 6th ed., ASM Press, Washington,D.C. USA ¹³Dowson, C. G., T. J. Tracey, and B. G. Spratt. 1994. Originand molecular epidemiology of penicillin-binding-protein- mediatedresistance to β-lactam antibiotics. Trends Molec. Microbiol.2: 361-366.¹⁴Jensen, L. B., N. Frimodt-Moller, F. M. Aarestrup. 1999. Presence oferm gene classes in Gram-positive bacteria of animal and human origin inDenmark. FEMS Microbiol. 170:151-158. ¹⁵Thal, L. A., and M. J. Zervos.1999. Occurrence and epidemiology of resistance to virginimycin andstreptrogramins. J. Antimicrob. Chemother. 43:171-176. ¹⁶Martinez J. L.,A. Alonso, J. M. Gomez-Gomez, and F. Baquero. 1998. Quinolone resistanceby mutations in chromosomal gyrase genes. Just the tip of the iceberg?J. Antimicrob. Chemother. 42:683-688 ¹⁷Cockerill III, F.R. 1999. Geneticmethods for assessing antimicrobial resistance. Antimicrob. Agents.Chemother. 43:199-212. ¹⁸Casadewall, B. and P. Courvalin. 1999Characterization of the vanD glycopeptide resistance gene cluster fromEnterococcus faecium BM 4339. J. Bacteriol. 181:3644-3648. ¹⁹Roberts,M.C. 1999. Genetic mobility and distribution of tetracycline resistancedeterminants. Ciba Found. Symp. 207:206-222. ²⁰Huovinen, P., L.Sundström, G. Swedberg, and O. Sköld. 1995. Trimethoprim and sulfonamideresistance. Antimicrob. Agent Chemother. 39:279-289.

TABLE 6 List of bacterial toxins selected for diagnostic purposes.Accession Organism Toxin number Actinobacillus Cytolethal distendingAF006830 actinomycetemcomitans toxin (cdtA, cdtB, cdtC) Leukotoxin(ltxA) M27399 Actinomyces Hemolysin (pyolysin) U84782 pyogenes AeromonasAerolysin (aerA) M16495 hydrophila Haemolysin (hlyA) U81555 Cytotonicenterotoxin (alt) L77573 Bacillus anthracis Anthrax toxin (cya) M23179Bacillus cereus Enterotoxin (bceT) D17312 AF192766, AF192767 Enterotoxichemolysin BL AJ237785 Non-haemolytic enterotoxins Y19005 A,B and C (nhe)Bacillus mycoides Hemolytic enterotoxin HBL AJ243150 to AJ243153Bacillus Hemolytic enterotoxin HBL AJ243154 to pseudomycoides AJ243156Bacteroides Enterotoxin (bftP) U67735 fragilis Matrix metalloprotease/S75941, enterotoxin (fragilysin) AF038459 Metalloprotease toxin-2 U90931AF081785 Metalloprotease toxin-3 AF056297 Bordetella Adenylate cyclaseZ37112, bronchiseptica hemolysin (cyaA) U22953 Dermonecrotic toxin (dnt)U59687 AB020025 Bordetella Pertussis toxin (S1 subunit, tox) AJ006151pertussis AJ006153 AJ006155 AJ006157 AJ006159 AJ007363 M14378, M16494AJ007364 M13223 X16347 Adenyl cyclase (cya) 18323 Dermonecrotic toxin(dnt) U10527 Campylobacter Cytolethal distending toxin U51121 jejuni(cdtA, cdtB, cdtC) Citrobacter Shiga-like toxin (slt-IIcA) X67514,freundii S53206 Clostridium Botulism toxin (BoNT) (A, X52066, botulinumB, E and F serotypes X52088 are neurotoxic for humans; X73423 the otherserotypes M30196 have not been considered) X70814 X70819 X71343 Z11934X70817 M81186 X70818 X70815 X62089 X62683 S76749 X81714 X70816 X70820X70281 L35496 M92906 Clostridium A toxin (enterotoxin) AB012304difficile (tcdA) (cdtA) AF053400 Y12616 X51797 X17194 M30307 B toxin(cytotoxin) Z23277 (toxB) (cdtB) X53138 Clostridium Alpha (phospholipaseL43545 perfringens C) (cpa) L43546 L43547 L43548 X13608 X17300 D10248Beta (dermonecrotic L13198 protein) (cpb) X83275 L77965 Enterotoxin(cpe) AJ000766 M98037 X81849 X71844 Y16009 Enterotoxin pseudogeneAF037328 (not expressed) AF037329 AF037330 Epsilon toxin (etxD) M80837M95206 X60694 Iota (Ia and Ib) X73562 Lambda (metalloprotease) D45904Theta (perfringolysin O) M36704 Clostridium sordellii Cytotoxin L X82638Clostridium tetani Tetanos toxin X06214 X04436 CorynebacteriumDiphtheriae toxin X00703 diphtheriae Corynebacterium Phospholipase CA21336 pseudotuberculosis Eikenella corrodens lysine decarboxylase(cadA) U89166 Enterobacter cloacae Shiga-like toxin II Z50754, U33502Enterococcus faecalis Cytolysin B (cylB) M38052 Escherichia coliHemolysin toxin (hlyA AF043471 (EHEC) and ehxA) X94129 X79839 X86087AB011549 AF074613

TABLE 7 Origin of the nucleic acids and/or sequences in the sequencelisting. SEQ Archaeal, bacterial, fungal ID NO. or parasitical speciesSource Gene* 1 Acinetobacter baumannii This patent tuf 2 Actinomycesmeyeri This patent tuf 3 Aerococcus viridans This patent tuf 4Achromobacter xylosoxidans This patent tuf subsp. denitrificans 5Anaerorhabdus furcosus This patent tuf 6 Bacillus anthracis This patenttuf 7 Bacillus cereus This patent tuf 8 Bacteroides distasonis Thispatent tuf 9 Enterococcus casseliflavus This patent tuf 10Staphylococcus saprophyticus This patent tuf 11 Bacteroides ovatus Thispatent tuf 12 Bartonella henselae This patent tuf 13 Bifidobacteriumadolescentis This patent tuf 14 Bifidobacterium dentium This patent tuf15 Brucella abortus This patent tuf 16 Burkholderia cepacia This patenttuf 17 Cedecea davisae This patent tuf 18 Cedecea neteri This patent tuf19 Cedecea lapagei This patent tuf 20 Chlamydia pneumoniae This patenttuf 21 Chlamydia psittaci This patent tuf 22 Chlamydia trachomatis Thispatent tuf 23 Chryseobacterium This patent tuf meningosepticum 24Citrobacter amalonaticus This patent tuf 25 Citrobacter braakii Thispatent tuf 26 Citrobacter koseri This patent tuf 27 Citrobacter farmeriThis patent tuf 28 Citrobacter freundii This patent tuf 29 Citrobactersedlakii This patent tuf 30 Citrobacter werkmanii This patent tuf 31Citrobacter youngae This patent tuf 32 Clostridium perfringens Thispatent tuf 33 Comamonas acidovorans This patent tuf 34 Corynebacteriumbovis This patent tuf 35 Corynebacterium cervicis This patent tuf 36Corynebacterium flavescens This patent tuf 37 Corynebacterium kutscheriThis patent tuf 38 Corynebacterium This patent tuf minutissimum 39Corynebacterium mycetoides This patent tuf 40 Corynebacterium Thispatent tuf pseudogenitalium 41 Corynebacterium renale This patent tuf 42Corynebacterium ulcerans This patent tuf 43 Corynebacterium urealyticumThis patent tuf 44 Corynebacterium xerosis This patent tuf 45 Coxiellaburnetii This patent tuf 46 Edwardsiella hoshinae This patent tuf 47Edwardsiella tarda This patent tuf 48 Eikenella corrodens This patenttuf 49 Enterobacter aerogenes This patent tuf 50 Enterobacteragglomerans This patent tuf 51 Enterobacter amnigenus This patent tuf 52Enterobacter asburiae This patent tuf 53 Enterobacter cancerogenus Thispatent tuf 54 Enterobacter cloacae This patent tuf 55 Enterobactergergoviae This patent tuf 56 Enterobacter hormaechei This patent tuf 57Enterobacter sakazakii This patent tuf 58 Enterococcus casseliflavusThis patent tuf 59 Enterococcus cecorum This patent tuf 60 Enterococcusdispar This patent tuf 61 Enterococcus durans This patent tuf 62Enterococcus faecalis This patent tuf 63 Enterococcus faecalis Thispatent tuf 64 Enterococcus faecium This patent tuf 65 Enterococcusflavescens This patent tuf 66 Enterococcus gallinarum This patent tuf 67Enterococcus hirae This patent tuf 68 Enterococcus mundtii This patenttuf 69 Enterococcus pseudoavium This patent tuf 70 Enterococcusraffinosus This patent tuf 71 Enterococcus saccharolyticus This patenttuf 72 Enterococcus solitarius This patent tuf 73 Enterococcuscasseliflavus This patent tuf (C) 74 Staphylococcus saprophyticus Thispatent unknown 75 Enterococcus flavescens This patent tuf (C) 76Enterococcus gallinarum This patent tuf (C) 77 Ehrlichia canis Thispatent tuf 78 Escherichia coli This patent tuf 79 Escherichia fergusoniiThis patent tuf 80 Escherichia hermannii This patent tuf 81 Escherichiavulneris This patent tuf 82 Eubacterium lentum This patent tuf 83Eubacterium nodatum This patent tuf 84 Ewingella americana This patenttuf 85 Francisella tularensis This patent tuf 86 Fusobacterium nucleatumThis patent tuf subsp. polymorphum 87 Gemella haemolysans This patenttuf 88 Gemella morbillorum This patent tuf 89 Haemophilus This patenttuf actinomycetemcomitans 90 Haemophilus aphrophilus This patent tuf 91Haemophilus ducreyi This patent tuf 92 Haemophilus haemolyticus Thispatent tuf 93 Haemophilus parahaemolyticus This patent tuf 94Haemophilus parainfluenzae This patent tuf 95 Haemophilus paraphrophilusThis patent tuf 96 Haemophilus segnis This patent tuf 97 Hafnia alveiThis patent tuf 98 Kingella kingae This patent tuf 99 Klebsiellaornithinolytica This patent tuf 100 Klebsiella oxytoca This patent tuf101 Klebsiella planticola This patent tuf 102 Klebsiella pneumoniae Thispatent tuf subsp. ozaenae 103 Klebsiella pneumoniae This patent tufpneumoniae 104 Klebsiella pneumoniae This patent tuf subsp.rhinoscleromatis 105 Kluyvera ascorbata This patent tuf 106 Kluyveracryocrescens This patent tuf 107 Kluyvera georgiana This patent tuf 108Lactobacillus casei This patent tuf subsp. casei 109 Lactococcus lactisThis patent tuf subsp. lactis 110 Leclercia adecarboxylata This patenttuf 111 Legionella micdadei This patent tuf 112 Legionella pneumophilaThis patent tuf subsp. pneumophila 113 Leminorella grimontii This patenttuf 114 Leminorella richardii This patent tuf 115 Leptospira interrogansThis patent tuf 116 Megamonas hypermegale This patent tuf 117Mitsuokella multacidus This patent tuf 118 Mobiluncus curtisii Thispatent tuf subsp. holmesii 119 Moellerella wisconsensis This patent tuf120 Moraxella catarrhalis This patent tuf 121 Morganella morganii Thispatent tuf subsp. morganii 122 Mycobacterium tuberculosis This patenttuf 123 Neisseria cinerea This patent tuf 124 Neisseria elongata Thispatent tuf subsp. elongata 125 Neisseria flavescens This patent tuf 126Neisseria gonorrhoeae This patent tuf 127 Neisseria lactamica Thispatent tuf 128 Neisseria meningitidis This patent tuf 129 Neisseriamucosa This patent tuf 130 Neisseria sicca This patent tuf 131 Neisseriasubflava This patent tuf 132 Neisseria weaveri This patent tuf 133Ochrobactrum anthropi This patent tuf 134 Pantoea agglomerans Thispatent tuf 135 Pantoea dispersa This patent tuf 136 Pasteurellamultocida This patent tuf 137 Peptostreptococcus This patent tufanaerobius 138 Peptostreptococcus This patent tuf asaccharolyticus 139Peptostreptococcus prevotii This patent tuf 140 Porphyromonas Thispatent tuf asaccharolytica 141 Porphyromonas gingivalis This patent tuf142 Pragia fontium This patent tuf 143 Prevotella melaninogenica Thispatent tuf 144 Prevotella oralis This patent tuf 145 Propionibacteriumacnes This patent tuf 146 Proteus mirabilis This patent tuf 147 Proteuspenneri This patent tuf 148 Proteus vulgaris This patent tuf 149Providencia alcalifaciens This patent tuf 150 Providencia rettgeri Thispatent tuf 151 Providencia rustigianii This patent tuf 152 Providenciastuartii This patent tuf 153 Pseudomonas aeruginosa This patent tuf 154Pseudomonas fluorescens This patent tuf 155 Pseudomonas stutzeri Thispatent tuf 156 Psychrobacter This patent tuf phenylpyruvicum 157Rahnella aquatilis This patent tuf 158 Salmonella choleraesuis Thispatent tuf subsp.arizonae 159 Salmonella choleraesuis This patent tufsubsp. choleraesuis serotype Choleraesuis 160 Salmonella choleraesuisThis patent tuf subsp. diarizonae 161 Salmonella choleraesuis Thispatent tuf subsp. choleraesuis serotype Heidelberg 162 Salmonellacholeraesuis This patent tuf subsp. houtenae 163 Salmonella choleraesuisThis patent tuf subsp. indica 164 Salmonella choleraesuis This patenttuf subsp. salamae 165 Salmonella choleraesuis This patent tuf subsp.choleraesuis serotype Typhi 166 Serratia fonticola This patent tuf 167Serratia liquefaciens This patent tuf 168 Serratia marcescens Thispatent tuf 169 Serratia odorifera This patent tuf 170 Serratiaplymuthica This patent tuf 171 Serratia rubidaea This patent tuf 172Shigella boydii This patent tuf 173 Shigella dysenteriae This patent tuf174 Shigella flexneri This patent tuf 175 Shigella sonnei This patenttuf 176 Staphylococcus aureus This patent tuf 177 Staphylococcus aureusThis patent tuf 178 Staphylococcus aureus This patent tuf 179Staphylococcus aureus This patent tuf 180 Staphylococcus aureus Thispatent tuf subsp. aureus 181 Staphylococcus auricularis This patent tuf182 Staphylococcus capitis This patent tuf subsp. capitis 183Macrococcus caseolyticus This patent tuf 184 Staphylococcus cohnii Thispatent tuf subsp. cohnii 185 Staphylococcus epidermidis This patent tuf186 Staphylococcus haemolyticus This patent tuf 187 Staphylococcuswarneri This patent tuf 188 Staphylococcus haemolyticus This patent tuf189 Staphylococcus haemolyticus This patent tuf 190 Staphylococcushaemolyticus This patent tuf 191 Staphylococcus hominis This patent tufsubsp. hominis 192 Staphylococcus warneri This patent tuf 193Staphylococcus hominis This patent tuf 194 Staphylococcus hominis Thispatent tuf 195 Staphylococcus hominis This patent tuf 196 Staphylococcushominis This patent tuf 197 Staphylococcus lugdunensis This patent tuf198 Staphylococcus saprophyticus This patent tuf 199 Staphylococcussaprophyticus This patent tuf 200 Staphylococcus saprophyticus Thispatent tuf 201 Staphylococcus sciuri This patent tuf subsp. sciuri 202Staphylococcus warneri This patent tuf 203 Staphylococcus warneri Thispatent tuf 204 Bifidobacterium longum This patent tuf 205Stenotrophomonas maltophilia This patent tuf 206 Streptococcusacidominimus This patent tuf 207 Streptococcus agalactiae This patenttuf 208 Streptococcus agalactiae This patent tuf 209 Streptococcusagalactiae This patent tuf 210 Streptococcus agalactiae This patent tuf211 Streptococcus anginosus This patent tuf 212 Streptococcus bovis Thispatent tuf 213 Streptococcus anginosus This patent tuf 214 Streptococcuscricetus This patent tuf 215 Streptococcus cristatus This patent tuf 216Streptococcus downei This patent tuf 217 Streptococcus dysgalactiae Thispatent tuf 218 Streptococcus equi subsp. equi This patent tuf 219Streptococcus ferus This patent tuf 220 Streptococcus gordonii Thispatent tuf 221 Streptococcus anginosus This patent tuf 222 Streptococcusmacacae This patent tuf 223 Streptococcus gordonii This patent tuf 224Streptococcus mutans This patent tuf 225 Streptococcus parasanguinisThis patent tuf 226 Streptococcus ratti This patent tuf 227Streptococcus sanguinis This patent tuf 228 Streptococcus sobrinus Thispatent tuf 229 Streptococcus suis This patent tuf 230 Streptococcusuberis This patent tuf 231 Streptococcus vestibularis This patent tuf232 Tatumella ptyseos This patent tuf 233 Trabulsiella guamensis Thispatent tuf 234 Veillonella parvula This patent tuf 235 Yersiniaenterocolitica This patent tuf 236 Yersinia frederiksenii This patenttuf 237 Yersinia intermedia This patent tuf 238 Yersinia pestis Thispatent tuf 239 Yersinia pseudotuberculosis This patent tuf 240 Yersiniarohdei This patent tuf 241 Yokenella regensburgei This patent tuf 242Achromobacter xylosoxidans This patent atpD subsp. denitrificans 243Acinetobacter baumannii This patent atpD 244 Acinetobacter lwoffii Thispatent atpD 245 Staphylococcus saprophyticus This patent atpD 246Alcaligenes faecalis This patent atpD subsp. faecalis 247 Bacillusanthracis This patent atpD 248 Bacillus cereus This patent atpD 249Bacteroides distasonis This patent atpD 250 Bacteroides ovatus Thispatent atpD 251 Leclercia adecarboxylata This patent atpD 252Stenotrophomonas maltophilia This patent atpD 253 Bartonella henselaeThis patent atpD 254 Bifidobacterium adolescentis This patent atpD 255Brucella abortus This patent atpD 256 Cedecea davisae This patent atpD257 Cedecea lapagei This patent atpD 258 Cedecea neteri This patent atpD259 Chryseobacterium This patent atpD meningosepticum 260 Citrobacteramalonaticus This patent atpD 261 Citrobacter braakii This patent atpD262 Citrobacter koseri This patent atpD 263 Citrobacter farmeri Thispatent atpD 264 Citrobacter freundii This patent atpD 265 Citrobacterkoseri This patent atpD 266 Citrobacter sedlakii This patent atpD 267Citrobacter werkmanii This patent atpD 268 Citrobacter youngae Thispatent atpD 269 Clostridium innocuum This patent atpD 270 Clostridiumperfringens This patent atpD 272 Corynebacterium diphtheriae This patentatpD 273 Corynebacterium This patent atpD pseudodiphtheriticum 274Corynebacterium ulcerans This patent atpD 275 Corynebacteriumurealyticum This patent atpD 276 Coxiella burnetii This patent atpD 277Edwardsiella hoshinae This patent atpD 278 Edwardsiella tarda Thispatent atpD 279 Eikenella corrodens This patent atpD 280 Enterobacteragglomerans This patent atpD 281 Enterobacter amnigenus This patent atpD282 Enterobacter asburiae This patent atpD 283 Enterobacter cancerogenusThis patent atpD 284 Enterobacter cloacae This patent atpD 285Enterobacter gergoviae This patent atpD 286 Enterobacter hormaechei Thispatent atpD 287 Enterobacter sakazakii This patent atpD 288 Enterococcusavium This patent atpD 289 Enterococcus casseliflavus This patent atpD290 Enterococcus durans This patent atpD 291 Enterococcus faecalis Thispatent atpD 292 Enterococcus faecium This patent atpD 293 Enterococcusgallinarum This patent atpD 294 Enterococcus saccharolyticus This patentatpD 295 Escherichia fergusonii This patent atpD 296 Escherichiahermannii This patent atpD 297 Escherichia vulneris This patent atpD 298Eubacterium lentum This patent atpD 299 Ewingella americana This patentatpD 300 Francisella tularensis This patent atpD 301 Fusobacteriumgonidiaformans This patent atpD 302 Fusobacterium necrophorum Thispatent atpD subsp. necrophorum 303 Fusobacterium nucleatum This patentatpD subsp. polymorphum 304 Gardnerella vaginalis This patent atpD 305Gemella haemolysans This patent atpD 306 Gemella morbillorum This patentatpD 307 Haemophilus ducreyi This patent atpD 308 Haemophilushaemolyticus This patent atpD 309 Haemophilus parahaemolyticus Thispatent atpD 310 Haemophilus parainfluenzae This patent atpD 311 Hafniaalvei This patent atpD 312 Kingella kingae This patent atpD 313Klebsiella pneumoniae subsp. This patent atpD ozaenae 314 Klebsiellaornithinolytica This patent atpD 315 Klebsiella oxytoca This patent atpD316 Klebsiella planticola This patent atpD 317 Klebsiella pneumoniaesubsp. This patent atpD pneumoniae 318 Kluyvera ascorbata This patentatpD 319 Kluyvera cryocrescens This patent atpD 320 Kluyvera georgianaThis patent atpD 321 Lactobacillus acidophilus This patent atpD 322Legionella pneumophila subsp. This patent atpD pneumophila 323Leminorella grimontii This patent atpD 324 Listeria monocytogenes Thispatent atpD 325 Micrococcus lylae This patent atpD 326 Moellerellawisconsensis This patent atpD 327 Moraxella catarrhalis This patent atpD328 Moraxella osloensis This patent atpD 329 Morganella morganii Thispatent atpD subsp. morganii 330 Pantoea agglomerans This patent atpD 331Pantoea dispersa This patent atpD 332 Pasteurella multocida This patentatpD 333 Pragia fontium This patent atpD 334 Proteus mirabilis Thispatent atpD 335 Proteus vulgaris This patent atpD 336 Providenciaalcalifaciens This patent atpD 337 Providencia rettgeri This patent atpD338 Providencia rustigianii This patent atpD 339 Providencia stuartiiThis patent atpD 340 Psychrobacter This patent atpD phenylpyruvicum 341Rahnella aquatilis This patent atpD 342 Salmonella choleraesuis Thispatent atpD subsp. arizonae 343 Salmonella choleraesuis This patent atpDsubsp. choleraesuis serotype Choleraesuis 344 Salmonella choleraesuisThis patent atpD subsp. diarizonae 345 Salmonella choleraesuis Thispatent atpD subsp. houtenae 346 Salmonella choleraesuis This patent atpDsubsp. indica 347 Salmonella choleraesuis This patent atpD subsp.choleraesuis serotype Paratyphi A 348 Salmonella choleraesuis Thispatent atpD subsp. choleraesuis serotype Paratyphi B 349 Salmonellacholeraesuis This patent atpD subsp. salamae 350 Salmonella choleraesuisThis patent atpD subsp. choleraesuis serotype Typhi 351 Salmonellacholeraesuis This patent atpD subsp. choleraesuis serotype Typhimurium352 Salmonella choleraesuis This patent atpD subsp. choleraesuisserotype Virchow 353 Serratia ficaria This patent atpD 354 Serratiafonticola This patent atpD 355 Serratia grimesii This patent atpD 356Serratia liquefaciens This patent atpD 357 Serratia marcescens Thispatent atpD 358 Serratia odorifera This patent atpD 359 Serratiaplymuthica This patent atpD 360 Serratia rubidaea This patent atpD 361Pseudomonas putida This patent atpD 362 Shigella boydii This patent atpD363 Shigella dysenteriae This patent atpD 364 Shigella flexneri Thispatent atpD 365 Shigella sonnei This patent atpD 366 Staphylococcusaureus This patent atpD 367 Staphylococcus auricularis This patent atpD368 Staphylococcus capitis This patent atpD subsp. capitis 369Staphylococcus cohnii This patent atpD subsp. cohnii 370 Staphylococcusepidermidis This patent atpD 371 Staphylococcus haemolyticus This patentatpD 372 Staphylococcus hominis subsp. This patent atpD hominis 373Staphylococcus hominis This patent atpD 374 Staphylococcus lugdunensisThis patent atpD 375 Staphylococcus saprophyticus This patent atpD 376Staphylococcus simulans This patent atpD 377 Staphylococcus warneri Thispatent atpD 378 Streptococcus acidominimus This patent atpD 379Streptococcus agalactiae This patent atpD 380 Streptococcus agalactiaeThis patent atpD 381 Streptococcus agalactiae This patent atpD 382Streptococcus agalactiae This patent atpD 383 Streptococcus agalactiaeThis patent atpD 384 Streptococcus dysgalactiae This patent atpD 385Streptococcus equi This patent atpD subsp. equi 386 Streptococcusanginosus This patent atpD 387 Streptococcus salivarius This patent atpD388 Streptococcus suis This patent atpD 389 Streptococcus uberis Thispatent atpD 390 Tatumella ptyseos This patent atpD 391 Trabulsiellaguamensis This patent atpD 392 Yersinia bercovieri This patent atpD 393Yersinia enterocolitica This patent atpD 394 Yersinia frederiksenii Thispatent atpD 395 Yersinia intermedia This patent atpD 396 Yersiniapseudotuberculosis This patent atpD 397 Yersinia rohdei This patent atpD398 Yokenella regensburgei This patent atpD 399 Yarrowia lipolytica Thispatent tuf (EF-1) 400 Absidia corymbifera This patent tuf (EF-1) 401Alternaria alternata This patent tuf (EF-1) 402 Aspergillus flavus Thispatent tuf (EF-1) 403 Aspergillus fumigatus This patent tuf (EF-1) 404Aspergillus fumigatus This patent tuf (EF-1) 405 Aspergillus niger Thispatent tuf (EF-1) 406 Blastoschizomyces capitatus This patent tuf (EF-1)407 Candida albicans This patent tuf (EF-1) 408 Candida albicans Thispatent tuf (EF-1) 409 Candida albicans This patent tuf (EF-1) 410Candida albicans This patent tuf (EF-1) 411 Candida albicans This patenttuf (EF-1) 412 Candida dubliniensis This patent tuf (EF-1) 413 Candidacatenulata This patent tuf (EF-1) 414 Candida dubliniensis This patenttuf (EF-1) 415 Candida dubliniensis This patent tuf (EF-1) 416 Candidafamata This patent tuf (EF-1) 417 Candida glabrata WO98/20157 tuf (EF-1)418 Candida guilliermondii This patent tuf (EF-1) 419 Candida haemuloniiThis patent tuf (EF-1) 420 Candida inconspicua This patent tuf (EF-1)421 Candida kefyr This patent tuf (EF-1) 422 Candida krusei WO98/20157tuf (EF-1) 423 Candida lambica This patent tuf (EF-1) 424 Candidalusitaniae This patent tuf (EF-1) 425 Candida norvegensis This patenttuf (EF-1) 426 Candida parapsilosis WO98/20157 tuf (EF-1) 427 Candidarugosa This patent tuf (EF-1) 428 Candida sphaerica This patent tuf(EF-1) 429 Candida tropicalis WO98/20157 tuf (EF-1) 430 Candida utilisThis patent tuf (EF-1) 431 Candida viswanathii This patent tuf (EF-1)432 Candida zeylanoides This patent tuf (EF-1) 433 Coccidioides immitisThis patent tuf (EF-1) 434 Cryptococcus albidus This patent tuf (EF-1)435 Exophiala jeanselmei This patent tuf (EF-1) 436 Fusarium oxysporumThis patent tuf (EF-1) 437 Geotrichum sp. This patent tuf (EF-1) 438Histoplasma capsulatum This patent tuf (EF-1) 439 Issatchenkiaorientalis This patent tuf (EF-1) Kudrjanzev 440 Malassezia furfur Thispatent tuf (EF-1) 441 Malassezia pachydermatis This patent tuf (EF-1)442 Malbranchea filamentosa This patent tuf (EF-1) 443 Metschnikowiapulcherrima This patent tuf (EF-1) 444 Paecilomyces lilacinus Thispatent tuf (EF-1) 445 Paracoccidioides brasiliensis This patent tuf(EF-1) 446 Penicillium marneffei This patent tuf (EF-1) 447 Pichiaanomala This patent tuf (EF-1) 448 Pichia anomala This patent tuf (EF-1)449 Pseudallescheria boydii This patent tuf (EF-1) 450 Rhizopus oryzaeThis patent tuf (EF-1) 451 Rhodotorula minuta This patent tuf (EF-1) 452Sporobolomyces salmonicolor This patent tuf (EF-1) 453 Sporothrixschenckii This patent tuf (EF-1) 454 Stephanoascus ciferrii This patenttuf (EF-1) 455 Trichophyton mentagrophytes This patent tuf (EF-1) 456Trichosporon cutaneum This patent tuf (EF-1) 457 Wangiella dermatitidisThis patent tuf (EF-1) 458 Aspergillus fumigatus This patent atpD 459Blastoschizomyces capitatus This patent atpD 460 Candida albicans Thispatent atpD 461 Candida dubliniensis This patent atpD 462 Candida famataThis patent atpD 463 Candida glabrata This patent atpD 464 Candidaguilliermondii This patent atpD 465 Candida haemulonii This patent atpD466 Candida inconspicua This patent atpD 467 Candida kefyr This patentatpD 468 Candida krusei This patent atpD 469 Candida lambica This patentatpD 470 Candida lusitaniae This patent atpD 471 Candida norvegensisThis patent atpD 472 Candida parapsilosis This patent atpD 473 Candidarugosa This patent atpD 474 Candida sphaerica This patent atpD 475Candida tropicalis This patent atpD 476 Candida utilis This patent atpD477 Candida viswanathii This patent atpD 478 Candida zeylanoides Thispatent atpD 479 Coccidioides immitis This patent atpD 480 Cryptococcusalbidus This patent atpD 481 Fusarium oxysporum This patent atpD 482Geotrichum sp. This patent atpD 483 Histoplasma capsulatum This patentatpD 484 Malassezia furfur This patent atpD 485 Malassezia pachydermatisThis patent atpD 486 Metschnikowia pulcherrima This patent atpD 487Penicillium marneffei This patent atpD 488 Pichia anomala This patentatpD 489 Pichia anomala This patent atpD 490 Rhodotorula minuta Thispatent atpD 491 Rhodotorula mucilaginosa This patent atpD 492Sporobolomyces salmonicolor This patent atpD 493 Sporothrix schenckiiThis patent atpD 494 Stephanoascus ciferrii This patent atpD 495Trichophyton mentagrophytes This patent atpD 496 Wangiella dermatitidisThis patent atpD 497 Yarrowia lipolytica This patent atpD 498Aspergillus fumigatus This patent tuf (M) 499 Blastoschizomycescapitatus This patent tuf (M) 500 Candida rugosa This patent tuf (M) 501Coccidioides immitis This patent tuf (M) 502 Fusarium oxysporum Thispatent tuf (M) 503 Histoplasma capsulatum This patent tuf (M) 504Paracoccidioides brasiliensis This patent tuf (M) 505 Penicilliummarneffei This patent tuf (M) 506 Pichia anomala This patent tuf (M) 507Trichophyton mentagrophytes This patent tuf (M) 508 Yarrowia lipolyticaThis patent tuf (M) 509 Babesia bigemina This patent tuf (EF-1) 510Babesia bovis This patent tuf (EF-1) 511 Crithidia fasciculata Thispatent tuf (EF-1) 512 Entamoeba histolytica This patent tuf (EF-1) 513Giardia lamblia This patent tuf (EF-1) 514 Leishmania tropica Thispatent tuf (EF-1) 515 Leishmania aethiopica This patent tuf (EF-1) 516Leishmania tropica This patent tuf (EF-1) 517 Leishmania donovani Thispatent tuf (EF-1) 518 Leishmania infantum This patent tuf (EF-1) 519Leishmania enriettii This patent tuf (EF-1) 520 Leishmania gerbilli Thispatent tuf (EF-1) 521 Leishmania hertigi This patent tuf (EF-1) 522Leishmania major This patent tuf (EF-1) 523 Leishmania amazonensis Thispatent tuf (EF-1) 524 Leishmania mexicana This patent tuf (EF-1) 525Leishmania tarentolae This patent tuf (EF-1) 526 Leishmania tropica Thispatent tuf (EF-1) 527 Neospora caninum This patent tuf (EF-1) 528Trichomonas vaginalis This patent tuf (EF-1) 529 Trypanosoma brucei Thispatent tuf (EF-1) subsp. brucei 530 Crithidia fasciculata This patentatpD 531 Leishmania tropica This patent atpD 532 Leishmania aethiopicaThis patent atpD 533 Leishmania donovani This patent atpD 534 Leishmaniainfantum This patent atpD 535 Leishmania gerbilli This patent atpD 536Leishmania hertigi This patent atpD 537 Leishmania major This patentatpD 538 Leishmania amazonensis This patent atpD 607 Enterococcusfaecalis WO98/20157 tuf 608 Enterococcus faecium WO98/20157 tuf 609Enterococcus gallinarum WO98/20157 tuf 610 Haemophilus influenzaeWO98/20157 tuf 611 Staphylococcus epidermidis WO98/20157 tuf 612Salmonella choleraesuis This patent tuf subsp. choleraesuis serotypeParatyphi A 613 Serratia ficaria This patent tuf 614 Enterococcusmalodoratus This patent tuf (C) 615 Enterococcus durans This patent tuf(C) 616 Enterococcus pseudoavium This patent tuf (C) 617 Enterococcusdispar This patent tuf (C) 618 Enterococcus avium This patent tuf (C)619 Saccharomyces cerevisiae Database tuf (M) 621 Enterococcus faeciumThis patent tuf (C) 622 Saccharomyces cerevisiae This patent tuf (EF-1)623 Cryptococcus neoformans This patent tuf (EF-1) 624 Candida albicansWO98/20157 tuf (EF-1) 662 Corynebacterium diphtheriae WO98/20157 tuf 663Candida catenulata This patent atpD 665 Saccharomyces cerevisiaeDatabase tuf (EF-1) 666 Saccharomyces cerevisiae Database atpD 667Trypanosoma cruzi This patent atpD 668 Corynebacterium glutamicumDatabase tuf 669 Escherichia coli Database atpD 670 Helicobacter pyloriDatabase atpD 671 Clostridium acetobutylicum Database atpD 672 Cytophagalytica Database atpD 673 Ehrlichia risticii This patent atpD 674 Vibriocholerae This patent atpD 675 Vibrio cholerae This patent tuf 676Leishmania enriettii This patent atpD 677 Babesia microti This patenttuf (EF-1) 678 Cryptococcus neoformans This patent atpD 679 Cryptococcusneoformans This patent atpD 680 Cunninghamella bertholletiae This patentatpD 684 Candida tropicalis Database atpD (V) 685 Enterococcus hiraeDatabase atpD (V) 686 Chlamydia pneumoniae Database atpD (V) 687Halobacterium salinarum Database atpD (V) 688 Homo sapiens Database atpD(V) 689 Plasmodium falciparum Database atpD (V) 690 Saccharomycescerevisiae Database atpD (V) 691 Schizosaccharomyces pombe Database atpD(V) 692 Trypanosoma congolense Database atpD (V) 693 Thermusthermophilus Database atpD (V) 698 Escherichia coli WO98/20157 tuf 709Borrelia burgdorferi Database atpD (V) 710 Treponema pallidum DatabaseatpD (V) 711 Chlamydia trachomatis Genome project atpD (V) 712Enterococcus faecalis Genome project atpD (V) 713 Methanosarcina barkeriDatabase atpD (V) 714 Methanococcus jannaschii Database atpD (V) 715Porphyromonas gingivalis Genome project atpD (V) 716 Streptococcuspneumoniae Genome project atpD (V) 717 Burkholderia mallei This patenttuf 718 Burkholderia pseudomallei This patent tuf 719 Clostridiumbeijerinckii This patent tuf 720 Clostridium innocuum This patent tuf721 Clostridium novyi This patent tuf 722 Clostridium septicum Thispatent tuf 723 Clostridium tertium This patent tuf 724 Clostridiumtetani This patent tuf 725 Enterococcus malodoratus This patent tuf 726Enterococcus sulfureus This patent tuf 727 Lactococcus garvieae Thispatent tuf 728 Mycoplasma pirum This patent tuf 729 Mycoplasmasalivarium This patent tuf 730 Neisseria polysaccharea This patent tuf731 Salmonella choleraesuis This patent tuf subsp. choleraesuis serotypeEnteritidis 732 Salmonella choleraesuis This patent tuf subsp.choleraesuis serotype Gallinarum 733 Salmonella choleraesuis This patenttuf subsp. choleraesuis serotype Paratyphi B 734 Salmonella choleraesuisThis patent tuf subsp. choleraesuis serotype Virchow 735 Serratiagrimesii This patent tuf 736 Clostridium difficile This patent tuf 737Burkholderia pseudomallei This patent atpD 738 Clostridium bifermentansThis patent atpD 739 Clostridium beijerinckii This patent atpD 740Clostridium difficile This patent atpD 741 Clostridium ramosum Thispatent atpD 742 Clostridium septicum This patent atpD 743 Clostridiumtertium This patent atpD 744 Comamonas acidovorans This patent atpD 745Klebsiella pneumoniae subsp. This patent atpD rhinoscleromatis 746Neisseria canis This patent atpD 747 Neisseria cinerea This patent atpD748 Neisseria cuniculi This patent atpD 749 Neisseria elongata Thispatent atpD subsp. elongata 750 Neisseria flavescens This patent atpD751 Neisseria gonorrhoeae This patent atpD 752 Neisseria gonorrhoeaeThis patent atpD 753 Neisseria lactamica This patent atpD 754 Neisseriameningitidis This patent atpD 755 Neisseria mucosa This patent atpD 756Neisseria subflava This patent atpD 757 Neisseria weaveri This patentatpD 758 Neisseria animalis This patent atpD 759 Proteus penneri Thispatent atpD 760 Salmonella choleraesuis This patent atpD subsp.choleraesuis serotype Enteritidis 761 Yersinia pestis This patent atpD762 Burkholderia mallei This patent atpD 763 Clostridium sordellii Thispatent atpD 764 Clostridium novyi This patent atpD 765 Clostridiumbotulinum This patent atpD 766 Clostridium histolyticum This patent atpD767 Peptostreptococcus prevotii This patent atpD 768 Absidia corymbiferaThis patent atpD 769 Alternaria alternata This patent atpD 770Aspergillus flavus This patent atpD 771 Mucor circinelloides This patentatpD 772 Piedraia hortai This patent atpD 773 Pseudallescheria boydiiThis patent atpD 774 Rhizopus oryzae This patent atpD 775 Scopulariopsiskoningii This patent atpD 776 Trichophyton mentagrophytes This patentatpD 777 Trichophyton tonsurans This patent atpD 778 Trichosporoncutaneum This patent atpD 779 Cladophialophora carrionii This patent tuf(EF-1) 780 Cunninghamella bertholletiae This patent tuf (EF-1) 781Curvularia lunata This patent tuf (EF-1) 782 Fonsecaea pedrosoi Thispatent tuf (EF-1) 783 Microsporum audouinii This patent tuf (EF-1) 784Mucor circinelloides This patent tuf (EF-1) 785 Phialophora verrucosaThis patent tuf (EF-1) 786 Saksenaea vasiformis This patent tuf (EF-1)787 Syncephalastrum racemosum This patent tuf (EF-1) 788 Trichophytontonsurans This patent tuf (EF-1) 789 Trichophyton mentagrophytes Thispatent tuf (EF-1) 790 Bipolaris hawaiiensis This patent tuf (EF-1) 791Aspergillus fumigatus This patent tuf (M) 792 Trichophytonmentagrophytes This patent tuf (M) 827 Clostridium novyi This patentatpD (V) 828 Clostridium difficile This patent atpD (V) 829 Clostridiumsepticum This patent atpD (V) 830 Clostridium botulinum This patent atpD(V) 831 Clostridium perfringens This patent atpD (V) 832 Clostridiumtetani This patent atpD (V) 833 Streptococcus pyogenes Database atpD (V)834 Babesia bovis This patent atpD (V) 835 Cryptosporidium parvum Thispatent atpD (V) 836 Leishmania infantum This patent atpD (V) 837Leishmania major This patent atpD (V) 838 Leishmania tarentolae Thispatent atpD (V) 839 Trypanosoma brucei This patent atpD (V) 840Trypanosoma cruzi This patent tuf (EF-1) 841 Trypanosoma cruzi Thispatent tuf (EF-1) 842 Trypanosoma cruzi This patent tuf (EF-1) 843Babesia bovis This patent tuf (M) 844 Leishmania aethiopica This patenttuf (M) 845 Leishmania amazonensis This patent tuf (M) 846 Leishmaniadonovani This patent tuf (M) 847 Leishmania infantum This patent tuf (M)848 Leishmania enriettii This patent tuf (M) 849 Leishmania gerbilliThis patent tuf (M) 850 Leishmania major This patent tuf (M) 851Leishmania mexicana This patent tuf (M) 852 Leishmania tarentolae Thispatent tuf (M) 853 Trypanosoma cruzi This patent tuf (M) 854 Trypanosomacruzi This patent tuf (M) 855 Trypanosoma cruzi This patent tuf (M) 856Babesia bigemina This patent atpD 857 Babesia bovis This patent atpD 858Babesia microti This patent atpD 859 Leishmania guyanensis This patentatpD 860 Leishmania mexicana This patent atpD 861 Leishmania tropicaThis patent atpD 862 Leishmania tropica This patent atpD 863 Bordetellapertussis Database tuf 864 Trypanosoma brucei brucei Database tuf (EF-1)865 Cryptosporidium parvum This patent tuf (EF-1) 866 Staphylococcussaprophyticus This patent atpD 867 Zoogloea ramigera This patent atpD868 Staphylococcus saprophyticus This patent tuf 869 Enterococcuscasseliflavus This patent tuf 870 Enterococcus casseliflavus This patenttuf 871 Enterococcus flavescens This patent tuf 872 Enterococcusgallinarum This patent tuf 873 Enterococcus gallinarum This patent tuf874 Staphylococcus haemolyticus This patent tuf 875 Staphylococcusepidermidis This patent tuf 876 Staphylococcus epidermidis This patenttuf 877 Staphylococcus epidermidis This patent tuf 878 Staphylococcusepidermidis This patent tuf 879 Enterococcus gallinarum This patent tuf880 Pseudomonas aeruginosa This patent tuf 881 Enterococcuscasseliflavus This patent tuf 882 Enterococcus casseliflavus This patenttuf 883 Enterococcus faecalis This patent tuf 884 Enterococcus faecalisThis patent tuf 885 Enterococcus faecium This patent tuf 886Enterococcus faecium This patent tuf 887 Zoogloea ramigera This patenttuf 888 Enterococcus faecalis This patent tuf 889 Aspergillus fumigatusThis patent atpD 890 Penicillium marneffei This patent atpD 891Paecilomyces lilacinus This patent atpD 892 Penicillium marneffei Thispatent atpD 893 Sporothrix schenckii This patent atpD 894 Malbrancheafilamentosa This patent atpD 895 Paecilomyces lilacinus This patent atpD896 Aspergillus niger This patent atpD 897 Aspergillus fumigatus Thispatent tuf (EF-1) 898 Penicillium marneffei This patent tuf (EF-1) 899Piedraia hortai This patent tuf (EF-1) 900 Paecilomyces lilacinus Thispatent tuf (EF-1) 901 Paracoccidioides brasiliensis This patent tuf(EF-1) 902 Sporothrix schenckii This patent tuf (EF-1) 903 Penicilliummarneffei This patent tuf (EF-1) 904 Curvularia lunata This patent tuf(M) 905 Aspergillus niger This patent tuf (M) 906 Bipolaris hawaiiensisThis patent tuf (M) 907 Aspergillus flavus This patent tuf (M) 908Alternaria alternata This patent tuf (M) 909 Penicillium marneffei Thispatent tuf (M) 910 Penicillium marneffei This patent tuf (M) 918Escherichia coli Database recA 929 Bacteroides fragilis This patent atpD(V) 930 Bacteroides distasonis This patent atpD (V) 931 Porphyromonasasaccharolytica This patent atpD (V) 932 Listeria monocytogenes Thispatent tuf 939 Saccharomyces cerevisiae Database recA (Rad51) 940Saccharomyces cerevisiae Database recA (Dmc1) 941 Cryptococcus humicolusThis patent atpD 942 Escherichia coli This patent atpD 943 Escherichiacoli This patent atpD 944 Escherichia coli This patent atpD 945Escherichia coli This patent atpD 946 Neisseria polysaccharea Thispatent atpD 947 Neisseria sicca This patent atpD 948 Streptococcus mitisThis patent atpD 949 Streptococcus mitis This patent atpD 950Streptococcus mitis This patent atpD 951 Streptococcus oralis Thispatent atpD 952 Streptococcus pneumoniae This patent atpD 953Streptococcus pneumoniae This patent atpD 954 Streptococcus pneumoniaeThis patent atpD 955 Streptococcus pneumoniae This patent atpD 956Babesia microti This patent atpD (V) 957 Entamoeba histolytica Thispatent atpD (V) 958 Fusobacterium nucleatum This patent atpD (V) subsp.polymorphum 959 Leishmania aethiopica This patent atpD (V) 960Leishmania tropica This patent atpD (V) 961 Leishmania guyanensis Thispatent atpD (V) 962 Leishmania donovani This patent atpD (V) 963Leishmania hertigi This patent atpD (V) 964 Leishmania mexicana Thispatent atpD (V) 965 Leishmania tropica This patent atpD (V) 966Peptostreptococcus anaerobius This patent atpD (V) 967 Bordetellapertussis This patent tuf 968 Bordetella pertussis This patent tuf 969Enterococcus columbae This patent tuf 970 Enterococcus flavescens Thispatent tuf 971 Streptococcus pneumoniae This patent tuf 972 Escherichiacoli This patent tuf 973 Escherichia coli This patent tuf 974Escherichia coli This patent tuf 975 Escherichia coli This patent tuf976 Mycobacterium avium This patent tuf 977 Streptococcus pneumoniaeThis patent tuf 978 Mycobacterium gordonae This patent tuf 979Streptococcus pneumoniae This patent tuf 980 Mycobacterium tuberculosisThis patent tuf 981 Staphylococcus warneri This patent tuf 982Streptococcus mitis This patent tuf 983 Streptococcus mitis This patenttuf 984 Streptococcus mitis This patent tuf 985 Streptococcus oralisThis patent tuf 986 Streptococcus pneumoniae This patent tuf 987Enterococcus hirae This patent tuf (C) 988 Enterococcus mundtii Thispatent tuf (C) 989 Enterococcus raffinosus This patent tuf (C) 990Bacillus anthracis This patent recA 991 Prevotella melaninogenica Thispatent recA 992 Enterococcus casseliflavus This patent tuf 993Streptococcus pyogenes Database speA 1002 Streptococcus pyogenesWO98/20157 tuf 1003 Bacillus cereus This patent recA 1004 Streptococcuspneumoniae This patent pbp1a 1005 Streptococcus pneumoniae This patentpbp1a 1006 Streptococcus pneumoniae This patent pbp1a 1007 Streptococcuspneumoniae This patent pbp1a 1008 Streptococcus pneumoniae This patentpbp1a 1009 Streptococcus pneumoniae This patent pbp1a 1010 Streptococcuspneumoniae This patent pbp1a 1011 Streptococcus pneumoniae This patentpbp1a 1012 Streptococcus pneumoniae This patent pbp1a 1013 Streptococcuspneumoniae This patent pbp1a 1014 Streptococcus pneumoniae This patentpbp1a 1015 Streptococcus pneumoniae This patent pbp1a 1016 Streptococcuspneumoniae This patent pbp1a 1017 Streptococcus pneumoniae This patentpbp1a 1018 Streptococcus pneumoniae This patent pbp1a 1019 Streptococcuspneumoniae This patent pbp2b 1020 Streptococcus pneumoniae This patentpbp2b 1021 Streptococcus pneumoniae This patent pbp2b 1022 Streptococcuspneumoniae This patent pbp2b 1023 Streptococcus pneumoniae This patentpbp2b 1024 Streptococcus pneumoniae This patent pbp2b 1025 Streptococcuspneumoniae This patent pbp2b 1026 Streptococcus pneumoniae This patentpbp2b 1027 Streptococcus pneumoniae This patent pbp2b 1028 Streptococcuspneumoniae This patent pbp2b 1029 Streptococcus pneumoniae This patentpbp2b 1030 Streptococcus pneumoniae This patent pbp2b 1031 Streptococcuspneumoniae This patent pbp2b 1032 Streptococcus pneumoniae This patentpbp2b 1033 Streptococcus pneumoniae This patent pbp2b 1034 Streptococcuspneumoniae This patent pbp2x 1035 Streptococcus pneumoniae This patentpbp2x 1036 Streptococcus pneumoniae This patent pbp2x 1037 Streptococcuspneumoniae This patent pbp2x 1038 Streptococcus pneumoniae This patentpbp2x 1039 Streptococcus pneumoniae This patent pbp2x 1040 Streptococcuspneumoniae This patent pbp2x 1041 Streptococcus pneumoniae This patentpbp2x 1042 Streptococcus pneumoniae This patent pbp2x 1043 Streptococcuspneumoniae This patent pbp2x 1044 Streptococcus pneumoniae This patentpbp2x 1045 Streptococcus pneumoniae This patent pbp2x 1046 Streptococcuspneumoniae This patent pbp2x 1047 Streptococcus pneumoniae This patentpbp2x 1048 Streptococcus pneumoniae This patent pbp2x 1049 Enterococcusfaecium This patent vanA 1050 Enterococcus gallinarum This patent vanA1051 Enterococcus faecium This patent vanA 1052 Enterococcus faeciumThis patent vanA 1053 Enterococcus faecium This patent vanA 1054Enterococcus faecalis This patent vanA 1055 Enterococcus gallinarum Thispatent vanA 1056 Enterococcus faecium This patent vanA 1057 Enterococcusflavescens This patent vanA 1058 Enterococcus gallinarum This patentvanC1 1059 Enterococcus gallinarum This patent vanC1 1060 Enterococcuscasseliflavus This patent vanC2 1061 Enterococcus casseliflavus Thispatent vanC2 1062 Enterococcus casseliflavus This patent vanC2 1063Enterococcus casseliflavus This patent vanC2 1064 Enterococcusflavescens This patent vanC3 1065 Enterococcus flavescens This patentvanC3 1066 Enterococcus flavescens This patent vanC3 1067 Enterococcusfaecium This patent vanXY 1068 Enterococcus faecium This patent vanXY1069 Enterococcus faecium This patent vanXY 1070 Enterococcus faecalisThis patent vanXY 1071 Enterococcus gallinarum This patent vanXY 1072Enterococcus faecium This patent vanXY 1073 Enterococcus flavescens Thispatent vanXY 1074 Enterococcus faecium This patent vanXY 1075Enterococcus gallinarum This patent vanXY 1076 Escherichia coli Databasestx₁ 1077 Escherichia coli Database stx₂ 1093 Staphylococcussaprophyticus This patent unknown 1117 Enterococcus faecium DatabasevanB 1138 Enterococcus gallinarum Database vanC1 1139 Enterococcusfaecium Database vanA 1140 Enterococcus casseliflavus Database vanC21141 Enterococcus faecium Database vanHAXY 1169 Streptococcus pneumoniaeDatabase pbp1a 1172 Streptococcus pneumoniae Database pbp2b 1173Streptococcus pneumoniae Database pbp2x 1178 Staphylococcus aureusDatabase mecA 1183 Streptococcus pneumoniae Database hexA 1184Streptococcus pneumoniae This patent hexA 1185 Streptococcus pneumoniaeThis patent hexA 1186 Streptococcus pneumoniae This patent hexA 1187Streptococcus pneumoniae This patent hexA 1188 Streptococcus oralis Thispatent hexA 1189 Streptococcus mitis This patent hexA 1190 Streptococcusmitis This patent hexA 1191 Streptococcus mitis This patent hexA 1198Staphylococcus saprophyticus This patent unknown 1215 Streptococcuspyogenes Database pcp 1230 Escherichia coli Database tuf (EF-G) 1242Enterococcus faecium Database ddl 1243 Enterococcus faecalis DatabasemtlF, mtlD 1244 Staphylococcus aureus This patent unknown subsp. aureus1245 Bacillus anthracis This patent atpD 1246 Bacillus mycoides Thispatent atpD 1247 Bacillus thuringiensis This patent atpD 1248 Bacillusthuringiensis This patent atpD 1249 Bacillus thuringiensis This patentatpD 1250 Bacillus weihenstephanensis This patent atpD 1251 Bacillusthuringiensis This patent atpD 1252 Bacillus thuringiensis This patentatpD 1253 Bacillus cereus This patent atpD 1254 Bacillus cereus Thispatent atpD 1255 Staphylococcus aureus This patent gyrA 1256 Bacillusweihenstephanensis This patent atpD 1257 Bacillus anthracis This patentatpD 1258 Bacillus thuringiensis This patent atpD 1259 Bacillus cereusThis patent atpD 1260 Bacillus cereus This patent atpD 1261 Bacillusthuringiensis This patent atpD 1262 Bacillus thuringiensis This patentatpD 1263 Bacillus thuringiensis This patent atpD 1264 Bacillusthuringiensis This patent atpD 1265 Bacillus anthracis This patent atpD1266 Paracoccidioides brasiliensis This patent tuf (EF-1) 1267Blastomyces dermatitidis This patent tuf (EF-1) 1268 Histoplasmacapsulatum This patent tuf (EF-1) 1269 Trichophyton rubrum This patenttuf (EF-1) 1270 Microsporum canis This patent tuf (EF-1) 1271Aspergillus versicolor This patent tuf (EF-1) 1272 Exophiala moniliaeThis patent tuf (EF-1) 1273 Hortaea werneckii This patent tuf (EF-1)1274 Fusarium solani This patent tuf (EF-1) 1275 Aureobasidium pullulansThis patent tuf (EF-1) 1276 Blastomyces dermatitidis This patent tuf(EF-1) 1277 Exophiala dermatitidis This patent tuf (EF-1) 1278 Fusariummoniliforme This patent tuf (EF-1) 1279 Aspergillus terreus This patenttuf (EF-1) 1280 Aspergillus fumigatus This patent tuf (EF-1) 1281Cryptococcus laurentii This patent tuf (EF-1) 1282 Emmonsia parva Thispatent tuf (EF-1) 1283 Fusarium solani This patent tuf (EF-1) 1284Sporothrix schenckii This patent tuf (EF-1) 1285 Aspergillus nidulansThis patent tuf (EF-1) 1286 Cladophialophora carrionii This patent tuf(EF-1) 1287 Exserohilum rostratum This patent tuf (EF-1) 1288 Bacillusthuringiensis This patent recA 1289 Bacillus thuringiensis This patentrecA 1299 Staphylococcus aureus Database gyrA 1300 Escherichia coliDatabase gyrA 1307 Staphylococcus aureus Database gyrB 1320 Escherichiacoli Database parC (grlA) 1321 Staphylococcus aureus Database parC(grlA) 1328 Staphylococcus aureus Database parE (grlB) 1348 unidentifiedbacterium Database aac2Ia 1351 Pseudomonas aeruginosa Database aac3Ib1356 Serratia marcescens Database aac3IIb 1361 Escherichia coli Databaseaac3IVa 1366 Enterobacter cloacae Database aac3VIa 1371 Citrobacterkoseri Database aac6Ia 1376 Serratia marcescens Database aac6Ic 1381Escherichia coli Database ant3Ia 1386 Staphylococcus aureus Databaseant4Ia 1391 Escherichia coli Database aph3Ia 1396 Escherichia coliDatabase aph3IIa 1401 Enterococcus faecalis Database aph3IIIa 1406Acinetobacter baumannii Database aph3VIa 1411 Pseudomonas aeruginosaDatabase blaCARB 1416 Klebsiella pneumoniae Database blaCMY-2 1423Escherichia coli Database blaCTX-M-1 1428 Salmonella choleraesuis subsp.Database blaCTX-M-2 choleraesuis serotype Typhimurium 1433 Pseudomonasaeruginosa Database blaIMP 1438 Escherichia coli Database blaOXA2 1439Pseudomonas aeruginosa Database blaOXA10 1442 Pseudomonas aeruginosaDatabase blaPER1 1445 Salmonella choleraesuis subsp. Database blaPER2choleraesuis serotype Typhimurium 1452 Staphylococcus epidermidisDatabase dfrA 1461 Escherichia coli Database dhfrIa 1470 Escherichiacoli Database dhfrIb 1475 Escherichia coli Database dhfrV 1480 Proteusmirabilis Database dhfrVI 1489 Escherichia coli Database dhfrVII 1494Escherichia coli Database dhfrVIII 1499 Escherichia coli Database dhfrIX1504 Escherichia coli Database dhfrXII 1507 Escherichia coli DatabasedhfrXIII 1512 Escherichia coli Database dhfrXV 1517 Escherichia coliDatabase dhfrXVII 1518 Acinetobacter lwoffii This patent fusA 1519Acinetobacter lwoffii This patent fusA-tuf spacer 1520 Acinetobacterlwoffii This patent tuf 1521 Haemophilus influenzae This patent fusA1522 Haemophilus influenzae This patent fusA-tuf spacer 1523 Haemophilusinfluenzae This patent tuf 1524 Proteus mirabilis This patent fusA 1525Proteus mirabilis This patent fusA-tuf spacer 1526 Proteus mirabilisThis patent tuf 1527 Campylobacter curvus This patent atpD 1530Escherichia coli Database ereA 1535 Escherichia coli Database ereB 1540Staphylococcus haemolyticus Database linA 1545 Enterococcus faeciumDatabase linB 1548 Streptococcus pyogenes Database mefA 1551Streptococcus pneumoniae Database mefE 1560 Escherichia coli DatabasemphA 1561 Candida albicans This patent tuf (EF-1) 1562 Candidadubliniensis This patent tuf (EF-1) 1563 Candida famata This patent tuf(EF-1) 1564 Candida glabrata This patent tuf (EF-1) 1565 Candidaguilliermondii This patent tuf (EF-1) 1566 Candida haemulonii Thispatent tuf (EF-1) 1567 Candida kefyr This patent tuf (EF-1) 1568 Candidalusitaniae This patent tuf (EF-1) 1569 Candida sphaerica This patent tuf(EF-1) 1570 Candida tropicalis This patent tuf (EF-1) 1571 Candidaviswanathii This patent tuf (EF-1) 1572 Alcaligenes faecalis This patenttuf subsp. faecalis 1573 Prevotella buccalis This patent tuf 1574Succinivibrio dextrinosolvens This patent tuf 1575 Tetragenococcushalophilus This patent tuf 1576 Campylobacter jejuni This patent atpDsubsp. jejuni 1577 Campylobacter rectus This patent atpD 1578Enterococcus casseliflavus This patent fusA 1579 Enterococcus gallinarumThis patent fusA 1580 Streptococcus mitis This patent fusA 1585Enterococcus faecium Database satG 1590 Cloning vector pFW16 DatabasetetM 1594 Enterococcus faecium Database vanD 1599 Enterococcus faecalisDatabase vanE 1600 Campylobacter jejuni This patent atpD subsp. doylei1601 Enterococcus sulfureus This patent atpD 1602 Enterococcussolitarius This patent atpD 1603 Campylobacter sputorum This patent atpDsubsp. sputorum 1604 Enterococcus pseudoavium This patent atpD 1607Klebsiella ornithinolytica This patent gyrA 1608 Klebsiella oxytoca Thispatent gyrA 1613 Staphylococcus aureus Database vatB 1618 Staphylococcuscohnii Database vatC 1623 Staphylococcus aureus Database vga 1628Staphylococcus aureus Database vgaB 1633 Staphylococcus aureus Databasevgb 1638 Aspergillus fumigatus This patent atpD 1639 Aspergillusfumigatus This patent atpD 1640 Bacillus mycoides This patent atpD 1641Bacillus mycoides This patent atpD 1642 Bacillus mycoides This patentatpD 1643 Bacillus pseudomycoides This patent atpD 1644 Bacilluspseudomycoides This patent atpD 1645 Budvicia aquatica This patent atpD1646 Buttiauxella agrestis This patent atpD 1647 Candida norvegica Thispatent atpD 1648 Streptococcus pneumoniae This patent pbp1a 1649Campylobacter lad This patent atpD 1650 Coccidioides immitis This patentatpD 1651 Emmonsia parva This patent atpD 1652 Erwinia amylovora Thispatent atpD 1653 Fonsecaea pedrosoi This patent atpD 1654 Fusariummoniliforme This patent atpD 1655 Klebsiella oxytoca This patent atpD1656 Microsporum audouinii This patent atpD 1657 Obesumbacterium proteusThis patent atpD 1658 Paracoccidioides brasiliensis This patent atpD1659 Plesiomonas shigelloides This patent atpD 1660 Shewanellaputrefaciens This patent atpD 1662 Campylobacter curvus This patent tuf1663 Campylobacter rectus This patent tuf 1664 Fonsecaea pedrosoi Thispatent tuf 1666 Microsporum audouinii This patent tuf 1667 Piedraiahortai This patent tuf 1668 Escherichia coli Database tuf 1669 Saksenaeavasiformis This patent tuf 1670 Trichophyton tonsurans This patent tuf1671 Enterobacter aerogenes This patent atpD 1672 Bordetella pertussisDatabase atpD 1673 Arcanobacterium This patent tuf haemolyticum 1674Butyrivibrio fibrisolvens This patent tuf 1675 Campylobacter jejuni Thispatent tuf subsp. doylei 1676 Campylobacter lari This patent tuf 1677Campylobacter sputorum This patent tuf subsp. sputorum 1678Campylobacter upsaliensis This patent tuf 1679 Globicatella sanguis Thispatent tuf 1680 Lactobacillus acidophilus This patent tuf 1681Leuconostoc mesenteroides This patent tuf subsp. dextranicum 1682Prevotella buccalis This patent tuf 1683 Ruminococcus bromii This patenttuf 1684 Paracoccidioides brasiliensis This patent atpD 1685 Candidanorvegica This patent tuf (EF-1) 1686 Aspergillus nidulans This patenttuf 1687 Aspergillus terreus This patent tuf 1688 Candida norvegica Thispatent tuf 1689 Candida parapsilosis This patent tuf 1702 Streptococcusgordonii WO98/20157 recA 1703 Streptococcus mutans WO98/20157 recA 1704Streptococcus pneumoniae WO98/20157 recA 1705 Streptococcus pyogenesWO98/20157 recA 1706 Streptococcus salivarius WO98/20157 recA subsp.thermophilus 1707 Escherichia coli WO98/20157 oxa 1708 Enterococcusfaecalis WO98/20157 blaZ 1709 Pseudomonas aeruginosa WO98/20157aac6′-IIa 1710 Staphylococcus aureus WO98/20157 ermA 1711 Escherichiacoli WO98/20157 ermB 1712 Staphylococcus aureus WO98/20157 ermC 1713Enterococcus faecalis WO98/20157 vanB 1714 Campylobacter jejuni Thispatent recA subsp. jejuni 1715 Abiotrophia adiacens WO98/20157 tuf 1716Abiotrophia defectiva WO98/20157 tuf 1717 Corynebacterium accolensWO98/20157 tuf 1718 Corynebacterium genitalium WO98/20157 tuf 1719Corynebacterium jeikeium WO98/20157 tuf 1720 Corynebacterium WO98/20157tuf pseudodiphtheriticum 1721 Corynebacterium striatum WO98/20157 tuf1722 Enterococcus avium WO98/20157 tuf 1723 Gardnerella vaginalisWO98/20157 tuf 1724 Listeria innocua WO98/20157 tuf 1725 Listeriaivanovii WO98/20157 tuf 1726 Listeria monocytogenes WO98/20157 tuf 1727Listeria seeligeri WO98/20157 tuf 1728 Staphylococcus aureus WO98/20157tuf 1729 Staphylococcus saprophyticus WO98/20157 tuf 1730 Staphylococcussimulans WO98/20157 tuf 1731 Streptococcus agalactiae WO98/20157 tuf1732 Streptococcus pneumoniae WO98/20157 tuf 1733 Streptococcussalivarius WO98/20157 tuf 1734 Agrobacterium radiobacter WO98/20157 tuf1735 Bacillus subtilis WO98/20157 tuf 1736 Bacteroides fragilisWO98/20157 tuf 1737 Borrelia burgdorferi WO98/20157 tuf 1738Brevibacterium linens WO98/20157 tuf 1739 Chlamydia trachomatisWO98/20157 tuf 1740 Fibrobacter succinogenes WO98/20157 tuf 1741Flavobacterium ferrugineum WO98/20157 tuf 1742 Helicobacter pyloriWO98/20157 tuf 1743 Micrococcus luteus WO98/20157 tuf 1744 Mycobacteriumtuberculosis WO98/20157 tuf 1745 Mycoplasma genitalium WO98/20157 tuf1746 Neisseria gonorrhoeae WO98/20157 tuf 1747 Rickettsia prowazekiiWO98/20157 tuf 1748 Salmonella choleraesuis WO98/20157 tuf subsp.choleraesuis serotype Typhimurium 1749 Shewanella putrefaciensWO98/20157 tuf 1750 Stigmatella aurantiaca WO98/20157 tuf 1751 Thiomonascuprina WO98/20157 tuf 1752 Treponema pallidum WO98/20157 tuf 1753Ureaplasma urealyticum WO98/20157 tuf 1754 Wolinella succinogenesWO98/20157 tuf 1755 Burkholderia cepacia WO98/20157 tuf 1756 Bacillusanthracis This patent recA 1757 Bacillus anthracis This patent recA 1758Bacillus cereus This patent recA 1759 Bacillus cereus This patent recA1760 Bacillus mycoides This patent recA 1761 Bacillus pseudomycoidesThis patent recA 1762 Bacillus thuringiensis This patent recA 1763Bacillus thuringiensis This patent recA 1764 Klebsiella oxytoca Thispatent gyrA 1765 Klebsiella pneumoniae This patent gyrA subsp. ozaenae1766 Klebsiella planticola This patent gyrA 1767 Klebsiella pneumoniaeThis patent gyrA 1768 Klebsiella pneumoniae This patent gyrA subsp.pneumoniae 1769 Klebsiella pneumoniae This patent gyrA subsp. pneumoniae1770 Klebsiella pneumoniae This patent gyrA subsp. rhinoscleromatis 1771Klebsiella terrigena This patent gyrA 1772 Legionella pneumophila Thispatent gyrA subsp. pneumophila 1773 Proteus mirabilis This patent gyrA1774 Providencia rettgeri This patent gyrA 1775 Proteus vulgaris Thispatent gyrA 1776 Yersinia enterocolitica This patent gyrA 1777Klebsiella oxytoca This patent parC (grlA) 1778 Klebsiella oxytoca Thispatent parC (grlA) 1779 Klebsiella pneumoniae This patent parC (grlA)subsp. ozaenae 1780 Klebsiella planticola This patent parC (grlA) 1781Klebsiella pneumoniae This patent parC (grlA) 1782 Klebsiella pneumoniaeThis patent parC (grlA) subsp. pneumoniae 1783 Klebsiella pneumoniaeThis patent parC (grlA) subsp. pneumoniae 1784 Klebsiella pneumoniaeThis patent parC (grlA) subsp. rhinoscleromatis 1785 Klebsiellaterrigena This patent parC (grIA) 1786 Bacillus cereus This patent fusA1787 Bacillus cereus This patent fusA 1788 Bacillus anthracis Thispatent fusA 1789 Bacillus cereus This patent fusA 1790 Bacillusanthracis This patent fusA 1791 Bacillus pseudomycoides This patent fusA1792 Bacillus cereus This patent fusA 1793 Bacillus anthracis Thispatent fusA 1794 Bacillus cereus This patent fusA 1795 Bacillusweihenstephanensis This patent fusA 1796 Bacillus mycoides This patentfusA 1797 Bacillus thuringiensis This patent fusA 1798 Bacillusweihenstephanensis This patent fusA-tuf spacer 1799 Bacillusthuringiensis This patent fusA-tuf spacer 1800 Bacillus anthracis Thispatent fusA-tuf spacer 1801 Bacillus pseudomycoides This patent fusA-tufspacer 1802 Bacillus anthracis This patent fusA-tuf spacer 1803 Bacilluscereus This patent fusA-tuf spacer 1804 Bacillus cereus This patentfusA-tuf spacer 1805 Bacillus mycoides This patent fusA-tuf spacer 1806Bacillus cereus This patent fusA-tuf spacer 1807 Bacillus cereus Thispatent fusA-tuf spacer 1808 Bacillus cereus This patent fusA-tuf spacer1809 Bacillus anthracis This patent fusA-tuf spacer 1810 Bacillusmycoides This patent tuf 1811 Bacillus thuringiensis This patent tuf1812 Bacillus cereus This patent tuf 1813 Bacillus weihenstephanensisThis patent tuf 1814 Bacillus anthracis This patent tuf 1815 Bacilluscereus This patent tuf 1816 Bacillus cereus This patent tuf 1817Bacillus anthracis This patent tuf 1818 Bacillus cereus This patent tuf1819 Bacillus anthracis This patent tuf 1820 Bacillus pseudomycoidesThis patent tuf 1821 Bacillus cereus This patent tuf 1822 Streptococcusoralis This patent fusA 1823 Budvicia aquatica This patent fusA 1824Buttiauxella agrestis This patent fusA 1825 Klebsiella oxytoca Thispatent fusA 1826 Plesiomonas shigelloides This patent fusA 1827Shewanella putrefaciens This patent fusA 1828 Obesumbacterium proteusThis patent fusA 1829 Klebsiella oxytoca This patent fusA-tuf spacer1830 Budvicia aquatica This patent fusA-tuf spacer 1831 Plesiomonasshigelloides This patent fusA-tuf spacer 1832 Obesumbacterium proteusThis patent fusA-tuf spacer 1833 Shewanella putrefaciens This patentfusA-tuf spacer 1834 Buttiauxella agrestis This patent fusA-tuf spacer1835 Campylobacter coli This patent tuf 1836 Campylobacter fetus Thispatent tuf subsp. fetus 1837 Campylobacter fetus This patent tuf subsp.venerealis 1838 Buttiauxella agrestis This patent tuf 1839 Klebsiellaoxytoca This patent tuf 1840 Plesiomonas shigelloides This patent tuf1841 Shewanella putrefaciens This patent tuf 1842 Obesumbacteriumproteus This patent tuf 1843 Budvicia aquatica This patent tuf 1844Abiotrophia adiacens This patent atpD 1845 Arcanobacterium This patentatpD haemolyticum 1846 Basidiobolus ranarum This patent atpD 1847Blastomyces dermatitidis This patent atpD 1848 Blastomyces dermatitidisThis patent atpD 1849 Campylobacter coli This patent atpD 1850Campylobacter fetus This patent atpD subsp. fetus 1851 Campylobacterfetus This patent atpD subsp. venerealis 1852 Campylobacter gracilisThis patent atpD 1853 Campylobacter jejuni This patent atpD subsp.jejuni 1854 Enterococcus cecorum This patent atpD 1855 Enterococcuscolumbae This patent atpD 1856 Enterococcus dispar This patent atpD 1857Enterococcus malodoratus This patent atpD 1858 Enterococcus mundtii Thispatent atpD 1859 Enterococcus raffinosus This patent atpD 1860Globicatella sanguis This patent atpD 1861 Lactococcus garvieae Thispatent atpD 1862 Lactococcus lactis This patent atpD 1863 Listeriaivanovii This patent atpD 1864 Succinivibrio dextrinosolvens This patentatpD 1865 Tetragenococcus halophilus This patent atpD 1866 Campylobacterfetus This patent recA subsp. fetus 1867 Campylobacter fetus This patentrecA subsp. venerealis 1868 Campylobacter jejuni This patent recA subsp.jejuni 1869 Enterococcus avium This patent recA 1870 Enterococcusfaecium This patent recA 1871 Listeria monocytogenes This patent recA1872 Streptococcus mitis This patent recA 1873 Streptococcus oralis Thispatent recA 1874 Aspergillus fumigatus This patent tuf (M) 1875Aspergillus versicolor This patent tuf (M) 1876 Basidiobolus ranarumThis patent tuf (M) 1877 Campylobacter gracilis This patent tuf 1878Campylobacter jejuni This patent tuf subsp. jejuni 1879 Coccidioidesimmitis This patent tuf (M) 1880 Erwinia amylovora This patent tuf 1881Salmonella choleraesuis This patent tuf subsp. choleraesuis serotypeTyphimurium 1899 Klebsiella pneumoniae Database blaSHV 1900 Klebsiellapneumoniae Database blaSHV 1901 Escherichia coli Database blaSHV 1902Klebsiella pneumoniae Database blaSHV 1903 Klebsiella pneumoniaeDatabase blaSHV 1904 Escherichia coli Database blaSHV 1905 Pseudomonasaeruginosa Database blaSHV 1927 Neisseria meningitidis Database blaTEM1928 Escherichia coli Database blaTEM 1929 Klebsiella oxytoca DatabaseblaTEM 1930 Escherichia coli Database blaTEM 1931 Escherichia coliDatabase blaTEM 1932 Escherichia coli Database blaTEM 1933 Escherichiacoli Database blaTEM 1954 Klebsiella pneumoniae Database gyrA subsp.pneumoniae 1956 Candida inconspicua This patent tuf (M) 1957 Candidautilis This patent tuf (M) 1958 Candida zeylanoides This patent tuf (M)1959 Candida catenulata This patent tuf (M) 1960 Candida krusei Thispatent tuf (M) 1965 Plasmid pGS05 Database sulll 1970 Transposon Tn10Database tetB 1985 Cryptococcus neoformans Database tuf (EF-1) 1986Cryptococcus neoformans Database tuf (EF-1) 1987 Saccharomycescerevisiae Database tuf (EF-1) 1988 Saccharomyces cerevisiae Databasetuf (EF-1) 1989 Eremothecium gossypii Database tuf (EF-1) 1990Eremothecium gossypii Database tuf (EF-1) 1991 Aspergillus oryzaeDatabase tuf (EF-1) 1992 Aureobasidium pullulans Database tuf (EF-1)1993 Histoplasma capsulatum Database tuf (EF-1) 1994 Neurospora crassaDatabase tuf (EF-1) 1995 Podospora anserina Database tuf (EF-1) 1996Podospora curvicolla Database tuf (EF-1) 1997 Sordaria macrosporaDatabase tuf (EF-1) 1998 Trichoderma reesei Database tuf (EF-1) 2004Candida albicans Database tuf (M) 2005 Schizosaccharomyces pombeDatabase tuf (M) 2010 Klebsiella pneumoniae Database blaTEM 2011Klebsiella pneumoniae Database blaTEM 2013 Kluyvera ascorbata Thispatent gyrA 2014 Kluyvera georgiana This patent gyrA 2047 Streptococcuspneumoniae Database pbp1A 2048 Streptococcus pneumoniae Database pbp1A2049 Streptococcus pneumoniae Database pbp1A 2050 Streptococcuspneumoniae Database pbp1A 2051 Streptococcus pneumoniae Database pbp1A2052 Streptococcus pneumoniae Database pbp1A 2053 Streptococcuspneumoniae Database pbp1A 2054 Streptococcus pneumoniae Database gyrA2055 Streptococcus pneumoniae Database parC 2056 Streptococcuspneumoniae This patent pbp1A 2057 Streptococcus pneumoniae This patentpbp1A 2058 Streptococcus pneumoniae This patent pbp1A 2059 Streptococcuspneumoniae This patent pbp1A 2060 Streptococcus pneumoniae This patentpbp1A 2061 Streptococcus pneumoniae This patent pbp1A 2062 Streptococcuspneumoniae This patent pbp1A 2063 Streptococcus pneumoniae This patentpbp1A 2064 Streptococcus pneumoniae This patent pbp1A 2072 Mycobacteriumtuberculosis Database rpoB 2097 Mycoplasma pneumoniae Database tuf 2101Mycobacterium tuberculosis Database inhA 2105 Mycobacterium tuberculosisDatabase embB 2129 Clostridium difficile Database cdtA 2130 Clostridiumdifficile Database cdtB 2137 Pseudomonas putida Genome project tuf 2138Pseudomonas aeruginosa Genome project tuf 2139 Campylobacter jejuniDatabase atpD 2140 Streptococcus pneumoniae Database pbp1a 2144Staphylococcus aureus Database mupA 2147 Escherichia coli Database catI2150 Escherichia coli Database catII 2153 Shigella flexneri DatabasecatIII 2156 Clostridium perfringens Database catP 2159 Staphylococcusaureus Database cat 2162 Staphylococcus aureus Database cat 2165Salmonella typhimurium Database ppflo-like 2183 Alcaligenes faecalisThis patent tuf subsp. faecalis 2184 Campylobacter coli This patent fusA2185 Succinivibrio dextrinosolvens This patent tuf 2186 Tetragenococcushalophilus This patent tuf 2187 Campylobacter jejuni This patent fusAsubsp. jejuni 2188 Campylobacter jejuni This patent fusA subsp. jejuni2189 Leishmania guyanensis This patent atpD 2190 Trypanosoma bruceibrucei This patent atpD 2191 Aspergillus nidulans This patent atpD 2192Leishmania panamensis This patent atpD 2193 Aspergillus nidulans Thispatent tuf (M) 2194 Aureobasidium pullulans This patent tuf (M) 2195Emmonsia parva This patent tuf (M) 2196 Exserohilum rostratum Thispatent tuf (M) 2197 Fusarium moniliforme This patent tuf (M) 2198Fusarium solani This patent tuf (M) 2199 Histoplasma capsulatum Thispatent tuf (M) 2200 Kocuria kristinae This patent tuf 2201 Vibriomimicus This patent tuf 2202 Citrobacter freundii This patent recA 2203Clostridium botulinum This patent recA 2204 Francisella tularensis Thispatent recA 2205 Peptostreptococcus anaerobius This patent recA 2206Peptostreptococcus This patent recA asaccharolyticus 2207 Providenciastuartii This patent recA 2208 Salmonella choleraesuis This patent recAsubsp. choleraesuis serotype Paratyphi A 2209 Salmonella choleraesuisThis patent recA subsp. choleraesuis serotype Typhimurium 2210Staphylococcus saprophyticus This patent recA 2211 Yersiniapseudotuberculosis This patent recA 2212 Zoogloea ramigera This patentrecA 2214 Abiotrophia adiacens This patent fusA 2215 Acinetobacterbaumannii This patent fusA 2216 Actinomyces meyeri This patent fusA 2217Clostridium difficile This patent fusA 2218 Corynebacterium diphtheriaeThis patent fusA 2219 Enterobacter cloacae This patent fusA 2220Klebsiella pneumoniae This patent fusA subsp. pneumoniae 2221 Listeriamonocytogenes This patent fusA 2222 Mycobacterium avium This patent fusA2223 Mycobacterium gordonae This patent fusA 2224 Mycobacterium kansasiiThis patent fusA 2225 Mycobacterium terrae This patent fusA 2226Neisseria polysaccharea This patent fusA 2227 Staphylococcus epidermidisThis patent fusA 2228 Staphylococcus haemolyticus This patent fusA 2229Succinivibrio dextrinosolvens This patent fusA 2230 Tetragenococcushalophilus This patent fusA 2231 Veillonella parvula This patent fusA2232 Yersinia pseudotuberculosis This patent fusA 2233 Zoogloea ramigeraThis patent fusA 2234 Aeromonas hydrophila This patent fusA 2235Abiotrophia adiacens This patent fusA-tuf spacer 2236 Acinetobacterbaumannii This patent fusA-tuf spacer 2237 Actinomyces meyeri Thispatent fusA-tuf spacer 2238 Clostridium difficile This patent fusA-tufspacer 2239 Corynebacterium diphtheriae This patent fusA-tuf spacer 2240Enterobacter cloacae This patent fusA-tuf spacer 2241 Klebsiellapneumoniae This patent fusA-tuf spacer subsp. pneumoniae 2242 Listeriamonocytogenes This patent fusA-tuf spacer 2243 Mycobacterium avium Thispatent fusA-tuf spacer 2244 Mycobacterium gordonae This patent fusA-tufspacer 2245 Mycobacterium kansasii This patent fusA-tuf spacer 2246Mycobacterium terrae This patent fusA-tuf spacer 2247 Neisseriapolysaccharea This patent fusA-tuf spacer 2248 Staphylococcusepidermidis This patent fusA-tuf spacer 2249 Staphylococcus haemolyticusThis patent fusA-tuf spacer 2255 Abiotrophia adiacens This patent tuf2256 Acinetobacter baumannii This patent tuf 2257 Actinomyces meyeriThis patent tuf 2258 Clostridium difficile This patent tuf 2259Corynebacterium diphtheriae This patent tuf 2260 Enterobacter cloacaeThis patent tuf 2261 Klebsiella pneumoniae This patent tuf subsp.pneumoniae 2262 Listeria monocytogenes This patent tuf 2263Mycobacterium avium This patent tuf 2264 Mycobacterium gordonae Thispatent tuf 2265 Mycobacterium kansasii This patent tuf 2266Mycobacterium terrae This patent tuf 2267 Neisseria polysaccharea Thispatent tuf 2268 Staphylococcus epidermidis This patent tuf 2269Staphylococcus haemolyticus This patent tuf 2270 Aeromonas hydrophilaThis patent tuf 2271 Bilophila wadsworthia This patent tuf 2272Brevundimonas diminuta This patent tuf 2273 Streptococcus mitis Thispatent pbp1a 2274 Streptococcus mitis This patent pbp1a 2275Streptococcus mitis This patent pbp1a 2276 Streptococcus oralis Thispatent pbp1a 2277 Escherichia coli This patent gyrA 2278 Escherichiacoli This patent gyrA 2279 Escherichia coli This patent gyrA 2280Escherichia coli This patent gyrA 2288 Enterococcus faecium Database ddl2293 Enterococcus faecium Database vanA 2296 Enterococcus faecalisDatabase vanB *tuf indicates tuf sequences, tuf (C) indicates tufsequences divergent from main (usually A and B) copies of the elongationfactor-Tu, tuf (EF-1) indicates tuf sequences of the eukaryotic type(elongation factor 1α), tuf (M) indicates tuf sequences from organellar(mostly mitochondrial) origin. fusA indicates fusA sequences; fusA-tufspacer indicates the intergenic region between fusA and tuf. atpDindicates atpD sequences of the F-type, atpD (V) indicates atpDsequences of the V-type. recA indicates recA sequences, recA(Rad51)indicates rad51 sequences or homologs and recA(Dmc1) indicates dmc1sequences or homologs.

TABLE 8 Bacterial species used to test the specificity of theStreptococcus agalactiae-specific amplification primers derived from tufsequences. Strain Reference number Streptococcus acidominimus ATCC 51726Streptococcus agalactiae ATCC 12403 Streptococcus agalactiae ATCC 12973Streptococcus agalactiae ATCC 13813 Streptococcus agalactiae ATCC 27591Streptococcus agalactiae CDCs 1073 Streptococcus anginosus ATCC 27335Streptococcus anginosus ATCC 33397 Streptococcus bovis ATCC 33317Streptococcus anginosus ATCC 27823 Streptococcus cricetus ATCC 19642Streptococcus cristatus ATCC 51100 Streptococcus downei ATCC 33748Streptococcus dysgalactiae ATCC 43078 Streptococcus equi subsp. equiATCC 9528  Streptococcus ferus ATCC 33477 Streptococcus gordonii ATCC10558 Streptococcus macacae ATCC 35911 Streptococcus mitis ATCC 49456Streptococcus mutans ATCC 25175 Streptococcus oralis ATCC 35037Streptococcus parasanguinis ATCC 15912 Streptococcus parauberis DSM 6631Streptococcus pneumoniae ATCC 27336 Streptococcus pyogenes ATCC 19615Streptococcus ratti ATCC 19645 Streptococcus salivarius ATCC 7073 Streptococcus sanguinis ATCC 10556 Streptococcus sobrinus ATCC 27352Streptococcus suis ATCC 43765 Streptococcus uberis ATCC 19436Streptococcus vestubularis ATCC 49124 Bacteroides caccae ATCC 43185Bacteroides vulgatus ATCC 8482  Bacteroides fragilis ATCC 25285 Candidaalbicans ATCC 11006 Clostridium innoculum ATCC 14501 Clostridium ramosumATCC 25582 Lactobacillus casei subsp. casei ATCC 393  Clostridiumsepticum ATCC 12464 Corynebacterium cervicis NCTC 10604 Corynebacteriumgenitalium ATCC 33031 Corynebacterium urealyticum ATCC 43042Enterococcus faecalis ATCC 29212 Enterococcus faecium ATCC 19434Eubacterium lentum ATCC 43055 Eubacterium nodutum ATCC 33099 Gardnerellavaginalis ATCC 14018 Lactobacillus acidophilus ATCC 4356  Lactobacilluscrispatus ATCC 33820 Lactobacillus gasseri ATCC 33323 Lactobacillusjohnsonii ATCC 33200 Lactococcus lactis subsp. lactis ATCC 19435Lactococcus lactis subsp. lactis ATCC 11454 Listeria innocua ATCC 33090Micrococcus luteus ATCC 9341  Escherichia coli ATCC 25922 Micrococcuslylae ATCC 27566 Porphyromonas asaccharolytica ATCC 25260 Prevotellacorporis ATCC 33547 Prevotella melanogenica ATCC 25845 Staphylococcusaureus ATCC 13301 Staphylococcus epidermidis ATCC 14990 Staphylococcussaprophyticus ATCC 15305

TABLE 9 Bacterial species used to test the specificity of theStreptococcus agalactiae-specific amplification primers derived fromatpD sequences. Strain Reference number Streptococcus acidominimus ATCC51726 Streptococcus agalactiae ATCC 12400 Streptococcus agalactiae ATCC12403 Streptococcus agalactiae ATCC 12973 Streptococcus agalactiae ATCC13813 Streptococcus agalactiae ATCC 27591 Streptococcus agalactiaeCDCs-1073 Streptococcus anginosus ATCC 27335 Streptococcus anginosusATCC 27823 Streptococcus bovis ATCC 33317 Streptococcus cricetus ATCC19642 Streptococcus cristatus ATCC 51100 Streptococcus downei ATCC 33748Streptococcus dysgalactiae ATCC 43078 Streptococcus equi subsp. equiATCC 9528 Streptococcus ferus ATCC 33477 Streptococcus gordonii ATCC10558 Streptococcus macacae ATCC 35911 Streptococcus mitis ATCC 49456Streptococcus mutans ATCC 25175 Streptococcus oralis ATCC 35037Streptococcus parasanguinis ATCC 15912 Streptococcus parauberis DSM 6631Streptococcus pneumoniae ATCC 27336 Streptococcus pyogenes ATCC 19615Streptococcus ratti ATCC 19645 Streptococcus salivarius ATCC 7073 Streptococcus sanguinis ATCC 10556 Streptococcus sobrinus ATCC 27352Streptococcus suis ATCC 43765 Streptococcus uberis ATCC 19436Streptococcus vestibularis ATCC 49124

TABLE 10 Bacterial species used to test the specificity of theEnterococcus-specific amplification primers derived from tuf sequences.Strain Reference number Gram-positive species (n = 74) Abiotrophiaadiacens ATCC 49176 Abiotrophia defectiva ATCC 49175 Bacillus cereusATCC 14579 Bacillus subtilis ATCC 27370 Bifidobacterium adolescentisATCC 27534 Bifidobacterium breve ATCC 15700 Bifidobacterium dentium ATCC27534 Bifidobacterium longum ATCC 15707 Clostridium perfringens ATCC3124  Clostridium septicum ATCC 12464 Corynebacterium aquaticus ATCC14665 Corynebacterium pseudodiphtheriticum ATCC 10700 Enterococcus aviumATCC 14025 Enterococcus casseliflavus ATCC 25788 Enterococcus cecorumATCC 43199 Enterococcus columbae ATCC 51263 Enterococcus dispar ATCC51266 Enterococcus durans ATCC 19432 Enterococcus faecalis ATCC 29212Enterococcus faecium ATCC 19434 Enterococcus flavescens ATCC 49996Enterococcus gallinarum ATCC 49573 Enterococcus hirae ATCC 8044 Enterococcus malodoratus ATCC 43197 Enterococcus mundtii ATCC 43186Enterococcus pseudoavium ATCC 49372 Enterococcus raffinosus ATCC 49427Enterococcus saccharolyticus ATCC 43076 Enterococcus solitarius ATCC49428 Enterococcus sulfureus ATCC 49903 Eubacterium lentum ATCC 49903Gemella haemolysans ATCC 10379 Gemella morbillorum ATCC 27842Lactobacillus acidophilus ATCC 4356  Leuconostoc mesenteroides ATCC19225 Listeria grayi ATCC 19120 Listeria grayi ATCC 19123 Listeriainnocua ATCC 33090 Listeria ivanovii ATCC 19119 Listeria monocytogenesATCC 15313 Listeria seeligeri ATCC 35967 Micrococcus luteus ATCC 9341 Pediococcus acidilacti ATCC 33314 Pediococcus pentosaceus ATCC 33316Peptococcus niger ATCC 27731 Peptostreptococcus anaerobius ATCC 27337Peptostreptococcus indolicus ATCC 29247 Peptostreptococcus micros ATCC33270 Propionibacterium acnes ATCC 6919  Staphylococcus aureus ATCC43300 Staphylococcus capitis ATCC 27840 Staphylococcus epidermidis ATCC14990 Staphylococcus haemolyticus ATCC 29970 Staphylococcus hominis ATCC27844 Staphylococcus lugdunensis ATCC 43809 Staphylococcus saprophyticusATCC 15305 Staphylococcus simulans ATCC 27848 Staphylococcus warneriATCC 27836 Streptococcus agalactiae ATCC 13813 Streptococcus anginosusATCC 33397 Streptococcus bovis ATCC 33317 Streptococcus constellatusATCC 27823 Streptococcus cristatus ATCC 51100 Streptococcus intermediusATCC 27335 Streptococcus mitis ATCC 49456 Streptococcus mitis ATCC 3639 Streptococcus mutans ATCC 27175 Streptococcus parasanguinis ATCC 15912Streptococcus pneumoniae ATCC 27736 Streptococcus pneumoniae ATCC 6303 Streptococcus pyogenes ATCC 19615 Streptococcus salivarius ATCC 7073 Streptococcus sanguinis ATCC 10556 Streptococcus suis ATCC 43765Gram-negative species (n = 39) Acidominococcus fermentans ATCC 2508 Acinetobacter baumannii ATCC 19606 Alcaligenes faecalis ATCC 8750 Anaerobiospirillum ATCC 29305 succiniproducens Anaerorhabdus furcosusATCC 25662 Bacteroides distasonis ATCC 8503  Bacteroidesthetaiotaomicron ATCC 29741 Bacteroides vulgatus ATCC 8482  Bordetellapertussis LSPQ 3702 Bulkholderia cepacia LSPQ 2217 Butyvibriofibrinosolvens ATCC 19171 Cardiobacterium hominis ATCC 15826 Citrobacterfreundii ATCC 8090  Desulfovibrio vulgaris ATCC 29579 Edwardsiellaetarda ATCC 15947 Enterobacter cloacae ATCC 13047 Escherichia coli ATCC25922 Fusobacterium russii ATCC 25533 Haemophilus influenzae ATCC 9007 Hafnia alvei ATCC 13337 Klebsiella oxytoca ATCC 13182 Meganomonashypermegas ATCC 25560 Mitsukoella multiacidus ATCC 27723 Moraxellacatarrhalis ATCC 43628 Morganella morganii ATCC 25830 Neisseriameningitidis ATCC 13077 Pasteurella aerogenes ATCC 27883 Proteusvulgaris ATCC 13315 Providencia alcalifaciens ATCC 9886  Providenciarettgeri ATCC 9250  Pseudomonas aeruginosa ATCC 27853 Salmonellatyphimurium ATCC 14028 Serratia marcescens ATCC 13880 Shigella flexneriATCC 12022 Shigella sonnei ATCC 29930 Succinivibrio dextrinosolvens ATCC19716 Tissierella praeacuta ATCC 25539 Veillonella parvuala ATCC 10790Yersinia enterocolitica ATCC 9610 

TABLE 11 Microbial species for which tuf and/or atpD and/or recAsequences are available in public databases. Species Strain Accessionnumber Coding gene* tuf sequences Bacteria Actinobacillusactinomycetemcomitans HK1651 Genome project² tuf Actinobacillusactinomycetemcomitans HK1651 Genome project² tuf (EF-G) Agrobacteriumtumefaciens X99673 tuf Agrobacterium tumefaciens X99673 tuf (EF-G)Agrobacterium tumefaciens X99674 tuf Anacystis nidulans PCC 6301 X17442tuf Aquifex aeolicus VF5 AE000669 tuf Aquifex aeolicus VF5 AE000669 tuf(EF-G) Aquifex pyrophilus Genome project² tuf (EF-G) Aquifex pyrophilusY15787 tuf Bacillus anthracis Ames Genome project² tuf Bacillusanthracis Ames Genome project² tuf (EF-G) Bacillus halodurans C-125AB017508 tuf Bacillus halodurans C-125 AB017508 tuf (EF-G) Bacillusstearothermophilus CCM 2184 AJ000260 tuf Bacillus subtilis 168 D64127tuf Bacillus subtilis 168 D64127 tuf (EF-G) Bacillus subtilis DSM 10Z99104 tuf Bacillus subtilis DSM 10 Z99104 tuf (EF-G) Bacteroidesforsythus ATCC 43037 AB035466 tuf Bacteroides fragilis DSM 1151 —¹ tufBordetella bronchiseptica RB50 Genome project² tuf Bordetella pertussisTohama 1 Genome project² tuf Bordetella pertussis Tohama 1 Genomeproject² tuf (EF-G) Borrelia burdorgferi B31 U78193 tuf Borreliaburgdorferi AE001155 tuf (EF-G) Brevibacterium linens DSM 20425 X76863tuf Buchnera aphidicola Ap Y12307 tuf Burkholderia pseudomallei K96243Genome project² tuf (EF-G) Campylobacter jejuni NCTC 11168 Y17167 tufCampylobacter jejuni NCTC 11168 CJ11168X2 tuf (EF-G) Chlamydiapneumoniae CWL029 AE001592 tuf Chlamydia pneumoniae CWL029 AE001639 tuf(EF-G) Chlamydia trachomatis M74221 tuf Chlamydia trachomatis D/UW-3/CXAE001317 tuf (EF-G) Chlamydia trachomatis D/UW-3/CX AE001305 tufChlamydia trachomatis F/IC-Cal-13 L22216 tuf Chlorobium vibrioforme DSM263 X77033 tuf Chloroflexus aurantiacus DSM 636 X76865 tuf Clostridiumacetobutylicum ATCC 824 Genome project² tuf Clostridium difficile 630Genome project² tuf Clostridium difficile 630 Genome project² tuf (EF-G)Corynebacterium diphtheriae NCTC 13129 Genome project² tufCorynebacterium diphtheriae NCTC 13129 Genome project² tuf (EF-G)Corynebacterium glutamicum ASO 19 X77034 tuf Corynebacterium glutamicumMJ-233 E09634 tuf Coxiella bumetii Nine Mile phase I AF136604 tufCytophaga lytica DSM 2039 X77035 tuf Deinococcus radiodurans R1 AE001891tuf (EF-G) Deinococcus radiodurans R1 AE180092 tuf Deinococcusradiodurans R1 AE002041 tuf Deinonema sp. —¹ tuf Eikenella corrodensATCC 23834 Z12610 tuf Eikenella corrodens ATCC 23834 Z12610 tuf (EF-G)Enterococcus faecalis Genome project² tuf (EF-G) Escherichia coli J01690tuf Escherichia coli J01717 tuf Escherichia coli X00415 tuf (EF-G)Escherichia coli X57091 tuf Escherichia coli K-12 MG1655 U000006 tufEscherichia coli K-12 MG1655 U000096 tuf Escherichia coli K-12 MG1655AE000410 tuf (EF-G) Fervidobacterium islandicum DSM 5733 Y15788 tufFibrobacter succinogenes S85 X76866 tuf Flavobacterium ferrigeneum DSM13524 X76867 tuf Flexistipes sinusarabici X59461 tuf Gloeobacterviolaceus PCC 7421 U09433 tuf Gloeothece sp. PCC 6501 U09434 tufHaemophilus actinomycetemcomitans HK1651 Genome project² tuf Haemophilusducreyi 35000 AF087414 tuf (EF-G) Haemophilus influenzae Rd U32739 tufHaemophilus influenzae Rd U32746 tuf Haemophilus influenzae Rd U32739tuf (EF-G) Helicobacter pylori 26695 AE000511 tuf Helicobacter pyloriJ99 AE001539 tuf (EF-G) Helicobacter pylori J99 AE001541 tufHerpetosiphon aurantiacus Hpga1 X76868 tuf Klebsiella pneumoniae M6H78578 Genome project² tuf Klebsiella pneumoniae M6H 78578 Genomeproject² tuf (EF-G) Lactobacillus paracasei E13922 tuf Legionellapneumophila Philadelphia-1 Genome project² tuf Leptospira interrogansAF115283 tuf Leptospira interrogans AF115283 tuf (EF-G) Micrococcusluteus IFO 3333 M17788 tuf (EF-G) Micrococcus luteus IFO 3333 M17788 tufMoraxella sp. TAC II 25 AJ249258 tuf Mycobacterium avium 104 Genomeproject² tuf Mycobacterium avium 104 Genome project² tuf (EF-G)Mycobacterium bovis AF2122/97 Genome project² tuf Mycobacterium bovisAF2122/97 Genome project² tuf (EF-G) Mycobacterium leprae L13276 tufMycobacterium leprae Z14314 tuf Mycobacterium leprae Z14314 tuf (EF-G)Mycobacterium leprae Thai 53 D13869 tuf Mycobacterium tuberculosisErdmann S40925 tuf Mycobacterium tuberculosis H37Rv AL021943 tuf (EF-G)Mycobacterium tuberculosis H37Rv Z84395 tuf Mycobacterium tuberculosisy42 AD000005 tuf Mycobacterium tuberculosis CSU#93 Genome project² tufMycobacterium tuberculosis CSU#93 Genome project² tuf (EF-G) Mycoplasmacapricolum PG-31 X16462 tuf Mycoplasma genitalium G37 U39732 tufMycoplasma genitalium G37 U39689 tuf (EF-G) Mycoplasma hominis X57136tuf Mycoplasma hominis PG21 M57675 tuf Mycoplasma pneumoniae M129AE000019 tuf Mycoplasma pneumoniae M129 AE000058 tuf (EF-G) Neisseriagonorrhoeae MS11 L36380 tuf Neisseria gonorrhoeae MS11 L36380 tuf (EF-G)Neisseria meningitidis Z2491 Genome project² tuf (EF-G) Neisseriameningitidis Z2491 Genome project² tuf Pasteurella multocida Pm70 Genomeproject² tuf Peptococcus niger DSM 20745 X76869 tuf Phormidium ectocarpiPCC 7375 U09443 tuf Planobispora rosea ATCC 53773 U67308 tufPlanobispora rosea ATCC 53733 X98830 tuf Planobispora rosea ATCC 53733X98830 tuf (EF-G) Plectonema boryanum PCC 73110 U09444 tuf Porphyromonasgingivalis W83 Genome project² tuf Porphyromonas gingivalis W83 Genomeproject² tuf (EF-G) Porphyromonas gingivalis FDC 381 AB035461 tufPorphyromonas gingivalis W83 AB035462 tuf Porphyromonas gingivalis SUNY1021 AB035463 tuf Porphyromonas gingivalis A7A1-28 AB035464 tufPorphyromonas gingivalis ATCC 33277 AB035465 tuf Porphyromonasgingivalis ATCC 33277 AB035471 tuf (EF-G) Prochlorothrix hollandicaU09445 tuf Pseudomonas aeruginosa PAO-1 Genome project² tuf Pseudomonasputida Genome project² tuf Rickettsia prowazekii Madrid E AJ235272 tufRickettsia prowazekii Madrid E AJ235270 tuf (EF-G) Rickettsia prowazekiiMadrid E Z54171 tuf (EF-G) Salmonella choleraesuis subsp. choleraesuisserotype Typhimurium X64591 tuf (EF-G) Salmonella choleraesuis subsp.choleraesuis serotype Typhimurium LT2 trpE91 X55116 tuf Salmonellacholeraesuis subsp. choleraesuis serotype Typhimurium LT2 trpE91 X55117tuf Serpulina hyodysenteriae B204 U51635 tuf Serratia marcescensAF058451 tuf Shewanella putrefaciens DSM 50426 —¹ tuf Shewanellaputrefaciens MR-1 Genome project² tuf Spirochaeta aurantia DSM 1902X76874 tuf Staphylococcus aureus AJ237696 tuf (EF-G) Staphylococcusaureus EMRSA-16 Genome project² tuf Staphylococcus aureus NCTC 8325Genome project² tuf Staphylococcus aureus COL Genome project² tufStaphylococcus aureus EMRSA-16 Genome project² tuf (EF-G) Stigmatellaaurantiaca DW4 X82820 tuf Stigmatella aurantiaca Sg a1 X76870 tufStreptococcus mutans GS-5 Kuramitsu U75481 tuf Streptococcus mutansUAB159 Genome project² tuf Streptococcus oralis NTCC 11427 P331701 tufStreptococcus pyogenes Genome project² tuf (EF-G) Streptococcus pyogenesM1-GAS Genome project² tuf Streptomyces aureofaciens ATCC 10762 AF007125tuf Streptomyces cinnamoneus Tue89 X98831 tuf Streptomyces coelicolorA3(2) AL031013 tuf (EF-G) Streptomyces coelicolor A3(2) X77039 tuf(EF-G) Streptomyces coelicolor M145 X77039 tuf Streptomyces collinus BSM40733 S79408 tuf Streptomyces netropsis Tu1063 AF153618 tuf Streptomycesramocissimus X67057 tuf Streptomyces ramocissimus X67058 tufStreptomyces ramocissimus X67057 tuf (EF-G) Synechococcus sp. PCC 6301X17442 tuf (EF-G) Synechococcus sp. PCC 6301 X17442 tuf Synechocystissp. PCC 6803 D90913 tuf (EF-G) Synechocystis sp. PCC 6803 D90913 tufSynechocystis sp. PCC 6803 X65159 tuf (EF-G) Taxeobacter occealus Myx2105 X77036 tuf Thermotoga maritima Genome project² tuf (EF-G)Thermotoga maritima M27479 tuf Thermus aquaticus EP 00276 X66322 tufThermus thermophilus HB8 X16278 tuf (EF-G) Thermus thermophilus HB8X05977 tuf Thermus thermophilus HB8 X06657 tuf Thiomonas cuprina DSM5495 U78300 tuf Thiomonas cuprina DSM 5495 U78300 tuf (EF-G) Thiomonascuprina Hoe5 X76871 tuf Treponema denticola Genome project² tufTreponema denticola Genome project² tuf (EF-G) Treponema pallidumAE001202 tuf Treponema pallidum AE001222 tuf (EF-G) Treponema pallidumAE001248 tuf (EF-G) Ureaplasma urealyticum ATCC 33697 Z34275 tufUreaplasma urealyticum serovar 3 biovar 1 AE002151 tuf Ureaplasmaurealyticum serovar 3 biovar 1 AE002151 tuf (EF-G) Vibrio choleraeN16961 Genome project² tuf Wolinella succinogenes DSM 1740 X76872 tufYersinia pestis CO-92 Genome project² tuf Yersinia pestis CO-92 Genomeproject² tuf (EF-G) Archaebacteria Archaeoglobus fulgidus Genomeproject² tuf (EF-G) Halobacterium marismortui X16677 tufMethanobacterium thermoautrophicum delta H AE000877 tuf Methanococcusjannaschii ATCC 43067 U67486 tuf Methanococcus vannielii X05698 tufPyrococcus abyssi Orsay AJ248285 tuf Thermoplasma acidophilum DSM 1728X53866 tuf Fungi Absidia glauca CBS 101.48 X54730 tuf (EF-1) Arxulaadeninivorans Ls3 Z47379 tuf (EF-1) Aspergillus oryzae KBN616 AB007770tuf (EF-1) Aureobasidium pullulans R106 U19723 tuf (EF-1) Candidaalbicans SC5314 Genome project² tuf (M) Candida albicans SC5314 M29934tuf (EF-1) Candida albicans SC5314 M29935 tuf (EF-1) Cryptococcusneoformans B3501 U81803 tuf (EF-1) Cryptococcus neoformans M1-106 U81804tuf (EF-1) Eremothecium gossypii ATCC 10895 X73978 tuf (EF-1)Eremothecium gossypii A29820 tuf (EF-1) Fusarium oxysporum NRRL 26037AF008498 tuf (EF-1) Histoplasma capsulatum 186AS U14100 tuf (EF-1)Podospora anserina X74799 tuf (EF-1) Podospora curvicolla VLV X96614 tuf(EF-1) Prototheca wickerhamii 263-11 AJ245645 tuf (EF-1) Pucciniagraminis race 32 X73529 tuf (EF-1) Reclinomonas americana ATCC 50394AF007261 tuf (M) Rhizomucor racemosus ATCC 1216B X17475 tuf (EF-1)Rhizomucor racemosus ATCC 1216B J02605 tuf (EF-1) Rhizomucor racemosusATCC 1216B X17476 tuf (EF-1) Rhodotorula mucilaginosa AF016239 tuf(EF-1) Saccharomyces cerevisiae K00428 tuf (M) Saccharomyces cerevisiaeM59369 tuf (EF-G) Saccharomyces cerevisiae X00779 tuf (EF-1)Saccharomyces cerevisiae X01638 tuf (EF-1) Saccharomyces cerevisiaeM10992 tuf (EF-1) Saccharomyces cerevisiae Alpha S288 X78993 tuf (EF-1)Saccharomyces cerevisiae M15666 tuf (EF-1) Saccharomyces cerevisiaeZ35987 tuf (EF-1) Saccharomyces cerevisiae S288C (AB972) U51033 tuf(EF-1) Schizophyllum commune 1-40 X94913 tuf (EF-1) Schizosaccharomycespombe 972h- AL021816 tuf (EF-1) Schizosaccharomyces pombe 972h- AL021813tuf (EF-1) Schizosaccharomyces pombe 972h- D82571 tuf (EF-1)Schizosaccharomyces pombe U42189 tuf (EF-1) Schizosaccharomyces pombePR745 D89112 tuf (EF-1) Sordaria macrospora OOO X96615 tuf (EF-1)Trichoderma reesei QM9414 Z23012 tuf (EF-1) Yarrowia lipolytica AF054510tuf (EF-1) Parasites Blastocystis hominis HE87-1 D64080 tuf (EF-1)Cryptosporidium parvum U69697 tuf (EF-1) Eimeria tenella LS18 A1755521tuf (EF-1) Entamoeba histolytica HM1:IMSS X83565 tuf (EF-1) Entamoebahistolytica NIH 200 M92073 tuf (EF-1) Giardia lamblia D14342 tuf (EF-1)Kentrophoros sp. AF056101 tuf (EF-1) Leishmania amazonensisIFLA/BR/67/PH8 M92653 tuf (EF-1) Leishmania braziliensis U72244 tuf(EF-1) Onchocerca volvulus M64333 tuf (EF-1) Porphyra purpurea AvonportU08844 tuf (EF-1) Plasmodium berghei ANKA AJ224150 tuf (EF-1) Plasmodiumfalciparum K1 X60488 tuf (EF-1) Plasmodium knowlesi line H AJ224153 tuf(EF-1) Toxoplasma gondii RH Y11431 tuf (EF-1) Trichomonas tenax ATCC30207 D78479 tuf (EF-1) Trypanosoma brucei LVH/75/ U10562 tuf (EF-1)USAMRU-K/18 Trypanosoma cruzi Y L76077 tuf (EF-1) Human and plantsArabidopsis thaliana Columbia X89227 tuf (EF-1) Glycine max CeresiaX89058 tuf (EF-1) Glycine max Ceresia Y15107 tuf (EF-1) Glycine maxCeresia Y15108 tuf (EF-1) Glycine max Maple Arrow X66062 tuf (EF-1) Homosapiens X03558 tuf (EF-1) Pyramimonas disomata AB008010 tuf atpDsequences Bacteria Acetobacterium woodi DSM 1030 U10505 atpDActinobacillus actinomycetemcomitans HK1651 Genome project² atpDBacillus anthracis Ames Genome project² atpD Bacillus firmus OF4 M60117atpD Bacillus megaterium QM B1551 M20255 atpD Bacillusstearothermophilus D38058 atpD Bacillus stearothermophilus IFO1035D38060 atpD Bacillus subtilis 168 Z28592 atpD Bacteroides fragilis DSM2151 M22247 atpD Bordetella bronchiseptica RB50 Genome project² atpDBordetella pertussis Tohama 1 Genome project² atpD Borrelia burgdorferiB31 AE001122 atpD (V) Burkholderia cepacia DSM50181 X76877 atpDBurkholderia pseudomallei K96243 Genome project² atpD Campylobacterjejuni NCTC 11168 CJ11168X1 atpD Chlamydia pneumoniae Genome project²atpD (V) Chlamydia trachomatis MoPn Genome project² atpD (V) Chlorobiumvibrioforme DSM 263 X76873 atpD Citrobacter freundii JEO503 AF037156atpD Clostridium acetobutylicum ATCC 824 Genome project² atpDClostridium acetobutylicum DSM 792 AF101055 atpD Clostridium difficile630 Genome project² atpD Corynebacterium diphtheriae NCTC13129 Genomeproject² atpD Corynebacterium glutamicum ASO 19 X76875 atpDCorynebacterium glutamicum MJ-233 E09634 atpD Cytophaga lytica DSM 2039M22535 atpD Enterobacter aerogenes DSM 30053 —³ atpD Enterococcusfaecalis V583 Genome project² atpD (V) Enterococcus hirae M90060 atpDEnterococcus hirae ATCC 9790 D17462 atpD (V) Escherichia coli J01594atpD Escherichia coli M25464 atpD Escherichia coli V00267 atpDEscherichia coli V00311 atpD Escherichia coli K12 MG1655 L10328 atpDFlavobacterium ferrugineum DSM 13524 —³ atpD Haemophilusactinomycetemcomitans Genome project² atpD Haemophilus influenzae RdU32730 atpD Helicobacter pylori NCTC 11638 AF004014 atpD Helicobacterpylori 26695 Genome project² atpD Helicobacter pylori J99 Genomeproject² atpD Klebsiella pneumoniae M6H 78578 Genome project² atpDLactobacillus casei DSM 20021 X64542 atpD Legionella pneumophilaPhiladelphia-1 Genome project² atpD Moorella thermoacetica ATCC 39073U64318 atpD Mycobacterium avium 104 Genome project² atpD Mycobacteriumbovis AF2122/97 Genome project² atpD Mycobacterium leprae U15186 atpDMycobacterium leprae Genome project² atpD Mycobacterium tuberculosisH37Rv Z73419 atpD Mycobacterium tuberculosis CSU#93 Genome project² atpDMycoplasma gallisepticum X64256 atpD Mycoplasma genitalium G37 U39725atpD Mycoplasma pneumoniae M129 U43738 atpD Neisseria gonorrhoeae FA1090 Genome project² atpD Neisseria meningitidis Z2491 Genome project²atpD Pasteurella multocida Pm70 Genome project² atpD Pectinatusfrisingensis DSM 20465 X64543 atpD Peptococcus niger DSM 20475 X76878atpD Pirellula marina IFAM 1313 X57204 atpD Porphyromonas gingivalis W83Genome project² atpD (V) Propionigenium modestum DSM 2376 X58461 atpDPseudomonas aeruginosa PAO1 Genome project² atpD Pseudomonas putidaGenome project² atpD Rhodobacter capsulatus B100 X99599 atpDRhodospirillum rubrum X02499 atpD Rickettsia prowazekii F-12 AF036246atpD Rickettsia prowazekii Madrid Genome project² atpD Ruminococcusalbus 7ATCC AB006151 atpD Salmonella bongori JEO4162 AF037155 atpDSalmonella bongori BR1859 AF037154 atpD Salmonella choleraesuis S83769AF037146 atpD subsp. arizonae Salmonella choleraesuis u24 AF037147 atpDsubsp. arizonae Salmonella choleraesuis subsp. K228 AF037140 atpDcholeraesuis serotype Dublin Salmonella choleraesuis subsp. K771AF037139 atpD choleraesuis serotype Dublin Salmonella choleraesuissubsp. Div36-86 AF037142 atpD choleraesuis serotype Infantis Salmonellacholeraesuis subsp. Div95-86 AF037143 atpD choleraesuis serotypeTennessee Salmonella choleraesuis subsp. LT2 AF037141 atpD choleraesuisserotype Typhimurium Salmonella choleraesuis DS210/89 AF037149 atpDsubsp. diarizonae Salmonella choleraesuis JEO307 AF037148 atpD subsp.diarizonae Salmonella choleraesuis S109671 AF037150 atpD subsp.diarizonae Salmonella choleraesuis S84366 AF037151 atpD subsp. houtenaeSalmonella choleraesuis S84098 AF037152 atpD subsp. houtenae Salmonellacholeraesuis BR2047 AF037153 atpD subsp. indica Salmonella choleraesuisNSC72 AF037144 atpD subsp. salamae Salmonella choleraesuis S114655AF037145 atpD subsp. salamae Shewanella putrefaciens MR-1 Genomeproject² atpD Staphylococcus aureus COL Genome project² atpD Stigmatellaaurantiaca Sga1 X76879 atpD Streptococcus bovis JB-1 AB009314 atpDStreptococcus mutans GS-5 U31170 atpD Streptococcus mutans UAB159 Genomeproject² atpD Streptococcus pneumoniae Type 4 Genome project² atpD (V)Streptococcus pneumoniae Type 4 Genome project² atpD Streptococcuspyogenes M1-GAS Genome project² atpD (V) Streptococcus pyogenes M1-GASGenome project² atpD Streptococcus sanguinis 10904 AF001955 atpDStreptomyces lividans 1326 Z22606 atpD Thermus thermophilus HB8 D63799atpD (V) Thiobacillus ferrooxidans ATCC 33020 M81087 atpD Treponemapallidum Nichols AE001228 atpD (V) Vibrio alginolyticus X16050 atpDVibrio cholerae N16961 Genome project² atpD Wolinella succinogenes DSM1470 X76880 atpD Yersinia enterocolitica NCTC 10460 AF037157 atpDYersinia pestis CO-92 Genome project² atpD Archaebacteria Archaeoglobusfulgidus DSM 4304 AE001023 atpD (V) Halobacterium salinarum S56356 atpD(V) Haloferax volcanii WR 340 X79516 atpD Methanococcus jannaschii DSM2661 U67477 atpD (V) Methanosarcina barkeri DSM 800 J04836 atpD (V)Fungi Candida albicans SC5314 Genome project² atpD Candida tropicalisM64984 atpD (V) Kluyveromyces lactis 2359/152 U37764 atpD Neurosporacrassa X53720 atpD Saccharomyces cerevisiae M12082 atpD Saccharomycescerevisiae X2180-1A J05409 atpD (V) Schizosaccharomyces pombe 972 h-S47814 atpD (V) Schizosaccharomyces pombe 972 h- M57956 atpD ParasitesGiardia lamblia WB U18938 atpD Plasmodium falciparum 3D7 L08200 atpD (V)Trypanosoma congolense IL3000 Z25814 atpD (V) Human and plants Homosapiens L09234 atpD (V) Homo sapiens M27132 atpD recA sequences BacteriaAcetobacter aceti no. 1023 S60630 recA Acetobacter altoacetigenes MH-24E05290 recA Acetobacter polyoxogenes NBI 1028 D13183 recA Acholeplasmalaidlawii 8195 M81465 recA Acidiphilium facilis ATCC 35904 D16538 recAAcidothermus cellulolyticus ATCC 43068 AJ006705 recA Acinetobactercalcoaceticus BD413/ADP1 L26100 recA Actinobacillusactinomycetemcomitans HK1651 Genome project² recA Aeromonas salmonicidaA449 U83688 recA Agrobacterium tumefaciens C58 L07902 recAAllochromatium vinosum AJ000677 recA Aquifex aeolicus VF5 AE000775 recAAquifex pyrophilus Kol5a L23135 recA Azotobacter vinelandii S96898 recABacillus stearothermophilus 10 Genome project² recA Bacillus subtilisPB1831 U87792 recA Bacillus subtilis 168 Z99112 recA Bacteroidesfragilis M63029 recA Bifidobacterium breve NCFB 2258 AF094756 recABlastochloris viridis DSM 133 AF022175 recA Bordetella pertussis 165X53457 recA Bordetella pertussis Tohama I Genome project² recA Borreliaburgdorferi Sh-2-82 U23457 recA Borrelia burgdorferi B31 AE001124 recABrevibacterium flavum MJ-233 E10390 recA Brucella abortus 2308 L00679recA Burkholderia cepacia ATCC 17616 U70431 recA Burkholderia cepaciaD90120 recA Burkholderia pseudomallei K96243 Genome project² recACampylobacter fetus subsp. fetus 23D AF020677 recA Campylobacter jejuni81-176 U03121 recA Campylobacter jejuni NCTC 11168 AL139079 recAChlamydia trachomatis L2 U16739 recA Chlamydia trachomatis D/UW-3/CXAE001335 recA Chlamydophila pneumoniae CWL029 AE001658 recA Chloroflexusaurantiacus J-10-fl AF037259 recA Clostridium acetobutylicum M94057 recAClostridium perfringens 13 U61497 recA Corynebacterium diphtheriaeNCTC13129 Genome project² recA Corynebacterium glutamicum AS019 U14965recA Corynebacterium pseudotuberculosis C231 U30387 recA Deinococcusradiodurans KD8301 AB005471 recA Deinococcus radiodurans R1 U01876 recAEnterobacter agglomerans 339 L03291 recA Enterococcus faecalis OGIXM81466 recA Erwinia carotovora X55554 recA Escherichia coli J01672 recAEscherichia coli X55552 recA Escherichia coli K-12 AE000354 recA Frankiaalni Arl3 AJ006707 recA Gluconobacter oxydans U21001 recA Haemophilusinfluenzae Rd U32687 recA Haemophilus influenzae Rd U32741 recAHaemophilus influenzae Rd L07529 recA Helicobacter pylori 69A Z35478recA Helicobacter pylori 26695 AE000536 recA Helicobacter pylori J99AE001453 recA Klebsiella pneumoniae M6H 78578 Genome project² recALactococcus lactis ML3 M88106 recA Legionella pneumophila X55453 recALeptospira biflexa serovar patoc U32625 recA Leptospira interrogansserovar pomona U29169 recA Magnetospirillum magnetotacticum MS-1 X17371recA Methylobacillus flagellatus MFK1 M35325 recA Methylomonas claraATCC 31226 X59514 recA Mycobacterium avium 104 Genome project² recAMycobacterium bovis AF122/97 Genome project² recA Mycobacterium lepraeX73822 recA Mycobacterium tuberculosis H37Rv X58485 recA Mycobacteriumtuberculosis CSU#93 Genome project² recA Mycoplasma genitalium G37U39717 recA Mycoplasma mycoides GM9 L22073 recA Mycoplasma pneumoniaeATCC 29342 MPAE000033 recA Mycoplasma pulmonis KD735 L22074 recAMyxococcus xanthus L40368 recA Myxococcus xanthus L40367 recA Neisseriaanimalis NCTC 10212 U57910 recA Neisseria cinerea LCDC 81-176 AJ223869recA Neisseria cinerea LNP 1646 U57906 recA Neisseria cinerea NCTC 10294AJ223871 recA Neisseria cinerea Vedros M601 AJ223870 recA Neisseriaelongate CCUG 2131 AJ223882 recA Neisseria elongate CCUG 4165A AJ223880recA Neisseria elongate NCTC 10660 AJ223881 recA Neisseria elongate NCTC11050 AJ223878 recA Neisseria elongate NHITCC 2376 AJ223877 recANeisseria elongate CCUG 4557 AJ223879 recA subsp. intermedia Neisseriaflava Bangor 9 AJ223873 recA Neisseria flavescens LNP 444 U57907 recANeisseria gonorrhoeae CH95 U57902 recA Neisseria gonorrhoeae FA19 X64842recA Neisseria gonorrhoeae MS11 X17374 recA Neisseria gonorrhoeae Genomeproject² recA Neisseria lactamica CCUC 7757 AJ223866 recA Neisserialactamica CCUG 7852 Y11819 recA Neisseria lactamica LCDC 77-143 Y11818recA Neisseria lactamica LCDC 80-111 AJ223864 recA Neisseria lactamicaLCDC 845 AJ223865 recA Neisseria lactamica NCTC 10617 U57905 recANeisseria lactamica NCTC 10618 AJ223863 recA Neisseria meningitidis44/46 X64849 recA Neisseria meningitidis Bangor 13 AJ223868 recANeisseria meningitidis HF116 X64848 recA Neisseria meningitidis HF130X64844 recA Neisseria meningitidis HF46 X64847 recA Neisseriameningitidis M470 X64850 recA Neisseria meningitidis N94II X64846 recANeisseria meningitidis NCTC 8249 AJ223867 recA Neisseria meningitidisP63 X64845 recA Neisseria meningitidis S3446 U57903 recA Neisseriameningitidis FAM18 Genome project² recA Neisseria mucosa LNP 405 U57908recA Neisseria mucosa Vedros M1801 AJ223875 recA Neisseria perflava CCUG17915 AJ223876 recA Neisseria perflava LCDC 85402 AJ223862 recANeisseria pharyngis var. flava NCTC 4590 U57909 recA Neisseriapolysaccharea CCUG 18031 Y11815 recA Neisseria polysaccharea CCUG 24845Y11816 recA Neisseria polysaccharea CCUG 24846 Y11814 recA Neisseriapolysaccharea INS MA 3008 Y11817 recA Neisseria polysaccharea NCTC 11858U57904 recA Neisseria sicca NRL 30016 AJ223872 recA Neisseria subflavaNRL 30017 AJ223874 recA Paracoccus denitrificans DSM 413 U59631 recAPasteurella multocida X99324 recA Porphyromonas gingivalis W83 U70054recA Prevotella ruminicola JCM 8958 U61227 recA Proteus mirabilis pG1300X14870 recA Proteus vulgaris X55555 recA Pseudomonas aeruginosa X05691recA Pseudomonas aeruginosa PAM 7 X52261 recA Pseudomonas aeruginosaPAO12 D13090 recA Pseudomonas fluorescens OE 28.3 M96558 recAPseudomonas putida L12684 recA Pseudomonas putida PpS145 U70864 recARhizobium leguminosarum VF39 X59956 recA biovar viciae Rhizobiumphaseoli CNPAF512 X62479 recA Rhodobacter capsulatus J50 X82183 recARhodobacter sphaeroides 2.4.1 X72705 recA Rhodopseudomonas palustris N 7D84467 recA Rickettsia prowazekii Madrid E AJ235273 recA Rickettsiaprowazekii Madrid E U01959 recA Serratia marcescens M22935 recA Shigellaflexneri X55553 recA Shigella sonnei KNIH104S AF101227 recASinorhizobium meliloti 2011 X59957 recA Staphylococcus aureus L25893recA Streptococcus gordonii Challis V288 L20574 recA Streptococcusmutans UA96 M81468 recA Streptococcus mutans GS-5 M61897 recAStreptococcus pneumoniae Z17307 recA Streptococcus pneumoniae R800Z34303 recA Streptococcus pyogenes NZ131 U21934 recA Streptococcuspyogenes D471 M81469 recA Streptococcus salivarius M94062 recA subsp.thermophilus Streptomyces ambofaciens DSM 40697 Z30324 recA Streptomycescoelicolor A3(2) AL020958 recA Streptomyces lividans TK24 X76076 recAStreptomyces rimosus R6 X94233 recA Streptomyces venezuelae ATCC10712U04837 recA Synechococcus sp. PR6 M29495 recA Synechocystis sp. PCC6803D90917 recA Thermotoga maritima L23425 recA Thermotoga maritima AE001823recA Thermus aquaticus L20095 recA Thermus thermophilus HB8 D17392 recAThiobacillus ferrooxidans M26933 recA Treponema denticola Genomeproject² recA Treponema pallidum Nichols AE001243 recA Vibrioanguillarum M80525 recA Vibrio cholerae 017 X71969 recA Vibrio cholerae2740-80 U10162 recA Vibrio cholerae 569B L42384 recA Vibrio choleraeM549 AF117881 recA Vibrio cholerae M553 AF117882 recA Vibrio choleraeM645 AF117883 recA Vibrio cholerae M793 AF117878 recA Vibrio choleraeM794 AF117880 recA Vibrio cholerae M967 AF117879 recA Xanthomonas citriXW47 AF006590 recA Xanthomonas oryzae AF013600 recA Xenorhabdus bovieniiT228/1 U87924 recA Xenorhabdus nematophilus AN6 AF127333 recA Yersiniapestis 231 X75336 recA Yersinia pestis CO-92 Genome project² recA Fungi,parasites, human and plants Anabaena variabilis ATCC 29413 M29680 recAArabidopsis thaliana U43652 recA (Rad51) Candida albicans U39808 recA(Dmc1) Coprinus cinereus Okayama-7 U21905 recA (Rad51) Emericellanidulans Z80341 recA (Rad51) Gallus gallus L09655 recA (Rad51) Homosapiens D13804 recA (Rad51) Homo sapiens D63882 recA (Dmc1) Leishmaniamajor Friedlin AF062379 recA (Rad51) Leishmania major Friedlin AF062380recA (Dmc1) Mus musculus D58419 recA (Dmc1) Neurospora crassa 74-OR23-1AD29638 recA (Rad51) Saccharomyces cerevisiae D10023 recA (Rad51)Schizosaccharomyces pombe Z22691 recA (Rad51) Schizosaccharomyces pombe972h- AL021817 recA (Dmc1) Tetrahymena thermophila PB9R AF064516 recA(Rad51) Trypanosoma brucei stock 427 Y13144 recA (Rad51) Ustilago maydisU62484 recA (Rad51) Xenopus laevis D38488 recA (Rad51) Xenopus laevisD38489 recA (Rad51) *tuf indicates tuf sequences, including tuf genes,fusA genes and fusA-tuf intergenic spacers. tuf (C) indicates tufsequences divergent from main (usually A and B) copies of the elongationfactor-Tu tuf (EF-1) indicates tuf sequences of the eukaryotic type(elongation factor 1α) tuf (M) indicates tuf sequences from organellar(mostly mitochondrial) origin atpD indicates atpD sequences of theF-type atpD (V) indicates atpD sequences of the V-Type recA indicatesrecA sequences recA (Rad51) indicates rad51 sequences or homologs recA(Dmc1) indicates dmc1 sequences or homologs ¹Nucleotides sequencespublished in Arch. Microbiol. 1990 153:241-247 ²These sequences are fromtheTIGR database (http://www.tigr.org/tdb/tdb.html) ³Nucleotidessequences published in FEMS Microbiology Letters 1988 50:101-106

TABLE 12 Bacterial species used to test the specificity of theStaphylococcus- specific amplification primers derived from tufsequences. Strain Reference number Staphylococcal species (n = 27)Staphylococcus arlettae ATCC 43957 Staphylococcus aureus ATCC 35844subsp. anaerobius Staphylococcus aureus ATCC 43300 subsp. aureusStaphylococcus auricularis ATCC 33753 Staphylococcus capitis ATCC 27840subsp. capitis Staphylococcus caprae ATCC 35538 Staphylococcus carnosusATCC 51365 Staphylococcus chromogenes ATCC 43764 Staphylococcus cohniiDSM 20260 subsp. urealyticum Staphylococcus delphini ATCC 49171Staphylococcus epidermidis ATCC 14990 Staphylococcus equorum ATCC 43958Staphylococcus felis ATCC 49168 Staphylococcus gallinarum ATCC 35539Staphylococcus haemolyticus ATCC 29970 Staphylococcus hominis ATCC 27844Staphylococcus hyicus ATCC 11249 Staphylococcus intermedius ATCC 29663Staphylococcus kloosis ATCC 43959 Staphylococcus lentus ATCC 29070Staphylococcus lugdunensis ATCC 43809 Staphylococcus saprophyticus ATCC15305 Staphylococcus schleiferi ATCC 49545 subsp. coagulansStaphylococcus sciuri ATCC 29060 subsp. sciuri Staphylococcus simulansATCC 27848 Staphylococcus warneri ATCC 27836 Staphylococcus xylosus ATCC29971 Gram-negative bacteria (n = 33) Acinetobacter baumannii ATCC 19606Bacteroides distasonis ATCC 8503 Bacteroides fragilis ATCC 25285Bulkholderia cepacia ATCC 25416 Bordetella pertussis ATCC 9797Citrobacter freundii ATCC 8090 Enterobacter aerogenes ATCC 13048Enterobacter cloacae ATCC 13047 Escherichia coli ATCC 25922 Haemophilusinfluenzae ATCC 8907 Haemophilus parahaemolyticus ATCC 10014 Haemophilusparainfluenzae ATCC 7901 Hafnia alvei ATCC 13337 Kingella indologenesATCC 25869 Klebsiella oxytoca ATCC 13182 Klebsiella pneumoniae ATCC13883 Moraxella catarrhalis ATCC 25240 Morganella morganii ATCC 25830Neisseria gonorrhoeae ATCC 35201 Neisseria meningitidis ATCC 13077Proteus mirabilis ATCC 25933 Proteus vulgaris ATCC 13315 Providenciarettgeri ATCC 9250 Providencia stuartii ATCC 29914 Pseudomonasaeruginosa ATCC 27853 Pseudomonas fluorencens ATCC 13525 Salmonellacholeraesuis ATCC 7001 Salmonella typhimurium ATCC 14028 Serratiamarcescens ATCC 8100 Shigella flexneri ATCC 12022 Shigella sonnei ATCC29930 Stenotrophomonas maltophilia ATCC 13843 Yersinia enterocoliticaATCC 9610 Other Gram-positive bacteria (n = 20) Bacillus subtilis ATCC27370 Enterococcus avium ATCC 14025 Enterococcus durans ATCC 19432Enterococcus faecalis ATCC 19433 Enterococcus faecium ATCC 19434Enterococcus flavescens ATCC 49996 Enterococcus gallinarum ATCC 49573Lactobacillus acidophilus ATCC 4356 Lactococcus lactis ATCC 11454Listeria innocua ATCC 33090 Listeria ivanovii ATCC 19119 Listeriamonocytogenes ATCC 15313 Macrococcus caseolyticus ATCC 13548Streptococcus agalactiae ATCC 13813 Streptococcus anginosus ATCC 33397Streptococcus bovis ATCC 33317 Streptococcus mutans ATCC 25175Streptococcus pneumoniae ATCC 6303 Streptococcus pyogenes ATCC 19615Streptococcus salivarius ATCC 7073

TABLE 13 Bacterial species used to test the specificity of thepenicillin-resistant Streptococcus pneumoniae assay. Strain Referencenumber Gram-positive species (n = 67) Abiotrophia adiacens ATCC 49175Abiotrophia defective ATCC 49176 Actinomyces pyogenes ATCC 19411Bacillus anthracis ATCC 4229 Bacillus cereus ATCC 14579 Bifidobacteriumbreve ATCC 15700 Clostridium difficile ATCC 9689 Enterococcus avium ATCC14025 Enterococcus casseliflavus ATCC 25788 Enterococcus dispar ATCC51266 Enterococcus durans ATCC 19432 Enterococcus faecalis ATCC 29212Enterococcus faecium ATCC 19434 Enterococcus flavescens ATCC 49996Enterococcus gallinarum ATCC 49573 Enterococcus hirae ATCC 8043Enterococcus mundtii ATCC 43186 Enterococcus raffinosus ATCC 49427Lactobacillus lactis ATCC 19435 Lactobacillus monocytogenes ATCC 15313Mobiluncus curtisii ATCC 35242 Peptococcus niger ATCC 27731Peptostreptococcus acones ATCC 6919 Peptostreptococcus anaerobius ATCC27337 Peptostreptococcus ATCC 2639 asaccharolyticus Peptostreptococcuslactolyticus ATCC 51172 Peptostreptococcus magnus ATCC 15794Peptostreptococcus prevotii ATCC 9321 Peptostreptococcus tetradius ATCC35098 Staphylococcus aureus ATCC 25923 Staphylococcus capitis ATCC 27840Staphylococcus epidermidis ATCC 14990 Staphylococcus haemolyticus ATCC29970 Staphylococcus hominis ATCC 27844 Staphylococcus lugdunensis ATCC43809 Staphylococcus saprophyticus ATCC 15305 Staphylococcus simulansATCC 27848 Staphylococcus. warneri ATCC 27836 Streptococcus acidominimusATCC 51726 Streptococcus agalactiae ATCC 12403 Streptococcus anginosusATCC 33397 Streptococcus bovis ATCC 33317 Streptococcus constellatusATCC 27823 Streptococcus cricetus ATCC 19624 Streptococcus cristatusATCC 51100 Streptococcus downei ATCC 33748 Streptococcus dysgalactiaeATCC 43078 Streptococcus equi ATCC 9528 Streptococcus ferus ATCC 33477Streptococcus gordonii ATCC 10558 Streptococcus intermedius ATCC 27335Streptococcus mitis ATCC 903 Streptococcus mitis LSPQ 2583 Streptococcusmitis ATCC 49456 Streptococcus mutans ATCC 27175 Streptococcus oralisATCC 10557 Streptococcus oralis ATCC 9811 Streptococcus oralis ATCC35037 Streptococcus parasanguinis ATCC 15912 Streptococcus parauberisATCC 6631 Streptococcus rattus ATCC 15912 Streptococcus salivarius ATCC7073 Streptococcus sanguinis ATCC10556 Streptococcus suis ATCC 43765Streptococcus uberis ATCC 19436 Streptococcus vestibularis ATCC 49124Gram-negative species (n = 33) Actinetobacter baumannii ATCC 19606Bordetella pertussis ATCC 9797 Citrobacter diversus ATCC 27028Citrobacter freundii ATCC 8090 Enterobacter aerogenes ATCC 13048Enterobacter agglomerans ATCC 27155 Enterobacter cloacae ATCC 13047Escherichia coli ATCC 25922 Haemophilus ducreyi ATCC 33940 Haemophilushaemolyticus ATCC 33390 Haemophilus influenzae ATCC 9007 Haemophilusparainfluenzae ATCC 7901 Hafnia alvei ATCC 13337 Klebsiella oxytoca ATCC13182 Klebsiella pneumoniae ATCC 13883 Moraxella atlantae ATCC 29525Moraxella catarrhalis ATCC 43628 Moraxella morganii ATCC 13077 Neisseriagonorrhoeae ATCC 35201 Neisseria meningitidis ATCC 13077 Proteusmirabilis ATCC 25933 Proteus vulgaris ATCC 13315 Providenciaalcalifaciens ATCC 9886 Providencia rettgeri ATCC 9250 Providenciarustigianii ATCC 33673 Providencia stuartii ATCC 33672 Pseudomonasaeruginosa ATCC 35554 Pseudomonas fluorescens ATCC 13525 Pseudomonasstutzeri ATCC 17588 Salmonella typhimurium ATCC 14028 Serratiamarcescens ATCC 13880 Shigella flexneri ATCC 12022 Yersinaenterocolitica ATCC 9610

TABLE 14 Bacterial species (n = 104) detected by the plateletcontaminants assay. Bold characters indicate the major bacterialcontaminants found in platelet concentrates. Abiotrophia adiacensAbiotrophia defectiva Acinetobacter baumannii Acinetobacter lwoffiAerococcus viridans Bacillus anthracis

Brucella abortus Burkholderia cepacia Citrobacter diversus Citrobacterfreundii Enterobacter aerogenes Enterobacter agglomerans

Enterococcus avium Enterococcus casseliflavus Enterococcus disparEnterococcus durans Enterococcus faecalis Enterococcus faeciumEnterococcus flavescens Enterococcus gallinarum Enterococcus mundtiiEnterococcus raffinosus Enterococcus solitarius

Gemella morbillorum Haemophilus ducreyi Haemophilus haemolyticusHaemophilus influenzae Haemophilus parahaemolyticus Haemophilusparainfluenzae Hafnia alvei Kingella kingae

Legionella pneumophila Megamonas hypermegale Moraxella atlantaeMoraxella catarrhalis Morganella morganii Neisseria gonorrheae Neisseriameningitidis Pasteurella aerogenes Pasteurella multocidaPeptostreptococcus magnus Proteus mirabilis Providencia alcalifaciensProvidencia rettgeri Providencia rustigianii Providencia stuartii

Pseudomonas fluorescens Pseudomonas stutzeri Salmonella bongori

Salmonella enteritidis Salmonella gallinarum Salmonella typhimuriumSerratia liquefaciens

Shigella flexneri Shigella sonnei

Staphylococcus capitis

Staphylococcus haemolyticus Staphylococcus hominis Staphylococcuslugdunensis Staphylococcus saprophyticus Staphylococcus simulansStaphylococcus warneri Stenotrophomonas maltophilia Streptococcusacidominimus

Streptococcus anginosus Streptococcus bovis Streptococcus constellatusStreptococcus cricetus Streptococcus cristatus Streptococcusdysgalactiae Streptococcus equi Streptococcus ferus Streptococcusgordonii Streptococcus intermedius Streptococcus macacae Streptococcusmitis

Streptococcus oralis Streptococcus parasanguinis Streptococcusparauberis Streptococcus pneumoniae

Streptococcus ratti

Streptococcus sobrinus Streptococcus uberis Streptococcus vestibularisVibrio cholerae Yersinia enterocolitica Yersinia pestis

TABLE 15 Microorganisms identified by commercial systems¹.   Abiotrophiaadiacens (Streptococcus adjacens) Abiotrophia defectiva (Streptococcusdefectivus) Achromobacter species Acidaminococcus fermentansAcinetobacter alcaligenes Acinetobacter anitratus Acinetobacterbaumannii Acinetobacter calcoaceticus Acinetobacter calcoaceticus biovaranitratus Acinetobacter calcoaceticus biovar lwoffi Acinetobactergenomospecies Acinetobacter haemolyticus Acinetobacter johnsoniiAcinetobacter junii Acinetobacter lwoffii Acinetobacter radioresistensAcinetobacter species Actinobacillus actinomycetemcomitansActinobacillus capsulatus Actinobacillus equuli Actinobacillus hominisActinobacillus lignieresii Actinobacillus pleuropneumoniaeActinobacillus species Actinobacillus suis Actinobacillus ureaeActinomyces bovis Actinomyces israelii Actinomyces meyeri Actinomycesnaeslundii Actinomyces neuii subsp. anitratus Actinomyces neuii subsp.neuii Actinomyces odontolyticus Actinomyces pyogenes Actinomycesradingae Actinomyces species Actinomyces turicensis Actinomyces viscosusAerococcus species Aerococcus viridans Aeromonas caviae Aeromonashydrophila Aeromonas hydrophila group Aeromonas jandaei Aeromonassalmonicida Aeromonas salmonicida subsp. achromogenes Aeromonassalmonicida subsp. masoucida Aeromonas salmonicida subsp. salmonicidaAeromonas schubertii Aeromonas sobria Aeromonas species Aeromonas trotaAeromonas veronii Aeromonas veronii biovar sobria Aeromonas veroniibiovar veronii Agrobacterium radiobacter Agrobacterium speciesAgrobacterium tumefaciens Alcaligenes denitrificans Alcaligenes faecalisAlcaligenes odorans Alcaligenes odorans (Alcaligenes faecalis)Alcaligenes species Alcaligenes xylosoxidans Alcaligenes xylosoxidanssubsp. denitrificans Alcaligenes xylosoxidans subsp. xylosoxidansAlloiococcus otitis Anaerobiospirillum succiniciproducens Anaerovibriolipolytica Arachnia propionica Arcanobacterium (Actinomyces) bernardiaeArcanobacterium (Actinomyces) pyogenes Arcanobacterium haemolyticumArcobacter cryaerophilus (Campylobacter cryaerophila) Arthrobacterglobiformis Arthrobacter species Arxiozyma telluris (Torulopsispintolopesii) Atopobium minutum (Lactobacillus minutus) Aureobacteriumspecies Bacillus amyloliquefaciens Bacillus anthracis Bacillus badiusBacillus cereus Bacillus circulans Bacillus coagulans Bacillus firmusBacillus lentus Bacillus licheniformis Bacillus megaterium Bacillusmycoides Bacillus pantothenticus Bacillus pumilus Bacillus speciesBacillus sphaericus Bacillus stearothermophilus Bacillus subtilisBacillus thuringiensis Bacteroides caccae Bacteroides capillosusBacteroides distasonis Bacteroides eggerthii Bacteroides fragilisBacteroides merdae Bacteroides ovatus Bacteroides species Bacteroidessplanchnicus Bacteroides stercoris Bacteroides thetaiotaomicronBacteroides uniformis Bacteroides ureolyticus (B. corrodens) Bacteroidesvulgatus Bergeyella (Weeksella) zoohelcum Bifidobacterium adolescentisBifidobacterium bifidum Bifidobacterium breve Bifidobacterium dentiumBifidobacterium infantis Bifidobacterium species Blastoschizomyces(Dipodascus) capitatus Bordetella avium Bordetella bronchisepticaBordetella parapertussis Bordetella pertussis Bordetella speciesBorrelia species Branhamella (Moraxella) catarrhalis Branhamella speciesBrevibacillus brevis Brevibacillus laterosporus Brevibacterium caseiBrevibacterium epidermidis Brevibacterium linens Brevibacterium speciesBrevundimonas (Pseudomonas) diminuta Brevundimonas (Pseudomonas)vesicularis Brevundimonas species Brochothrix thermosphacta Brucellaabortus Brucella canis Brucella melitensis Brucella ovis Brucellaspecies Brucella suis Budvicia aquatica Burkholderia (Pseudomonas)cepacia Burkholderia (Pseudomonas) gladioli Burkholderia (Pseudomonas)mallei Burkholderia (Pseudomonas) pseudomallei Burkholderia speciesButtiauxella agrestis Campylobacter coli Campylobacter concisusCampylobacter fetus Campylobacter fetus subsp. fetus Campylobacter fetussubsp. venerealis Campylobacter hyointestinalis Campylobacter jejunisubsp. doylei Campylobacter jejuni subsp. jejuni Campylobacter lariCampylobacter lari subsp. UPTC Campylobacter mucosalis Campylobacterspecies Campylobacter sputorum Campylobacter sputorum subsp. bubulusCampylobacter sputorum subsp. fecalis Campylobacter sputorum subsp.sputorum Campylobacter upsaliensis Candida (Clavispora) lusitaniaeCandida (Pichia) guilliermondii Candida (Torulopsis) glabrata Candidaalbicans Candida boidinii Candida catenulata Candida ciferrii Candidacolliculosa Candida conglobata Candida curvata (Cryptococcus curvatus)Candida dattila Candida dubliniensis Candida famata Candida globosaCandida hellenica Candida holmii Candida humicola Candida inconspicuaCandida intermedia Candida kefyr Candida krusei Candida lambica Candidamagnoliae Candida maris Candida melibiosica Candida membranaefaciensCandida norvegensis Candida norvegica Candida parapsilosis Candidaparatropicalis Candida pelliculosa Candida pseudotropicalis Candidapulcherrima Candida ravautii Candida rugosa Candida sake Candidasilvicola Candida species Candida sphaerica Candida stellatoidea Candidatenuis Candida tropicalis Candida utilis Candida valida Candida viniCandida viswanathii Candida zeylanoides Capnocytophaga gingivalisCapnocytophaga ochracea Capnocytophaga species Capnocytophaga sputigenaCardiobacterium hominis Carnobacterium divergens Carnobacteriumpiscicola CDC group ED-2 CDC group EF4 (Pasteurella sp.) CDC group EF-4ACDC group EF-4B CDC group EQ-Z CDC group HB-5 CDC group II K-2 CDC groupIV C-2 (Bordetella-like) CDC group M5 CDC group M6 Cedecea davisaeCedecea lapagei Cedecea neteri Cedecea species Cellulomonas (Oerskovia)turbata Cellulomonas species Chlamydia species Chromobacterium violaceumChryseobacterium (Flavobacterium) indologenes Chryseobacterium(Flavobacterium) meningosepticum Chryseobacterium gleum Chryseobacteriumspecies Chryseomonas indologenes Citeromyces matritensis Citrobacteramalonaticus Citrobacter braakii Citrobacter diversus Citrobacterfarmeri Citrobacter freundii Citrobacter freundii complex Citrobacterkoseri Citrobacter sedlakii Citrobacter species Citrobacter werkmaniiCitrobacter youngae Clostridium acetobutylicum Clostridium baratiClostridium beijerinckii Clostridium bifermentans Clostridium botulinumClostridium botulinum (NP) B&F Clostridium botulinum (NP) E Clostridiumbotulinum (P) A&H Clostridium botulinum (P) F Clostridium botulinum G1Clostridium botulinum G2 Clostridium butyricum Clostridium cadaverisClostridium chauvoei Clostridium clostridiiforme Clostridium difficileClostridium fallax Clostridium glycolicum Clostridium hastiformeClostridium histolyticum Clostridium innocuum Clostridium limosumClostridium novyi Clostridium novyi A Clostridium paraputrificumClostridium perfringens Clostridium putrificum Clostridium ramosumClostridium septicum Clostridium sordellii Clostridium speciesClostridium sphenoides Clostridium sporogenes Clostridium subterminaleClostridium tertium Clostridium tetani Clostridium tyrobutyricumComamonas (Pseudomonas) acidovorans Comamonas (Pseudomonas) testosteroniComamonas species Corynebacterium accolens Corynebacterium afermentansCorynebacterium amycolatum Corynebacterium aquaticum Corynebacteriumargentoratense Corynebacterium auris Corynebacterium bovisCorynebacterium coyleae Corynebacterium cystitidis Corynebacteriumdiphtheriae Corynebacterium diphtheriae biotype belfanti Corynebacteriumdiphtheriae biotype gravis Corynebacterium diphtheriae biotypeintermedius Corynebacterium diphtheriae biotype mitis Corynebacteriumflavescens Corynebacterium glucuronolyticum Corynebacteriumglucuronolyticum- seminale Corynebacterium group A Corynebacterium groupA-4 Corynebacterium group A-5 Corynebacterium group ANF Corynebacteriumgroup B Corynebacterium group B-3 Corynebacterium group FCorynebacterium group F-1 Corynebacterium group F-2 Corynebacteriumgroup G Corynebacterium group G-1 Corynebacterium group G-2Corynebacterium group I Corynebacterium group I-2 Corynebacteriumjeikeium (group JK) Corynebacterium kutscheri (C. murium)Corynebacterium macginleyi Corynebacterium minutissimum Corynebacteriumpilosum Corynebacterium propinquum Corynebacterium pseudodiphtheriticumCorynebacterium pseudotuberculosis Corynebacterium pyogenesCorynebacterium renale Corynebacterium renale group Corynebacteriumseminale Corynebacterium species Corynebacterium striatum (C. flavidum)Corynebacterium ulcerans Corynebacterium urealyticum (group D2)Corynebacterium xerosis Cryptococcus albidus Cryptococcus aterCryptococcus cereanus Cryptococcus gastricus Cryptococcus humicolusCryptococcus lactativorus Cryptococcus laurentii Cryptococcus luteolusCryptococcus melibiosum Cryptococcus neoformans Cryptococcus speciesCryptococcus terreus Cryptococcus uniguttulatus Debaryomyces hanseniiDebaryomyces marama Debaryomyces polymorphus Debaryomyces speciesDermabacter hominis Dermacoccus (Micrococcus) nishinomiyaensis Dietziaspecies Edwardsiella hoshinae Edwardsiella ictaluri Edwardsiella speciesEdwardsiella tarda Eikenella corrodens Empedobacter brevis(Flavobacterium breve) Enterobacter aerogenes Enterobacter agglomeransEnterobacter amnigenus Enterobacter amnigenus asburiae (CDC entericgroup 17) Enterobacter amnigenus biogroup 1 Enterobacter amnigenusbiogroup 2 Enterobacter asburiae Enterobacter cancerogenus Enterobactercloacae Enterobacter gergoviae Enterobacter hormaechei Enterobacterintermedius Enterobacter sakazakii Enterobacter species Enterobactertaylorae Enterobacter taylorae (CDC enteric group 19) Enterococcus(Streptococcus) cecorum Enterococcus (Streptococcus) faecalis (Group D)Enterococcus (Streptococcus) faecium (Group D) Enterococcus(Streptococcus) saccharolyticus Enterococcus avium (Group D)Enterococcus casseliflavus (Steptococcus faecium subsp. casseliflavus)Enterococcus durans (Streptococcus faecium subsp. durans) (Group D)Enterococcus gallinarum Enterococcus hirae Enterococcus malodoratusEnterococcus mundtii Enterococcus raffinosus Enterococcus speciesErwinia amylovora Erwinia carotovora Erwinia carotovora subsp.atroseptica Erwinia carotovora subsp. betavasculorum Erwinia carotovorasubsp. carotovora Erwinia chrysanthemi Erwinia cypripedii Erwiniamallotivora Erwinia nigrifluens Erwinia quercina Erwinia rhaponticiErwinia rubrifaciens Erwinia salicis Erwinia species Erysipelothrixrhusiopathiae Erysipelothrix species Escherichia blattae Escherichiacoli Escherichia coli A-D Escherichia coli O157:H7 Escherichiafergusonii Escherichia hermannii Escherichia species Escherichiavulneris Eubacterium aerofaciens Eubacterium alactolyticum Eubacteriumlentum Eubacterium limosum Eubacterium species Ewingella americanaFilobasidiella neoformans Filobasidium floriforme Filobasidiumuniguttulatum Flavimonas oryzihabitans Flavobacterium gleumFlavobacterium indologenes Flavobacterium odoratum Flavobacteriumspecies Francisella novicida Francisella philomiragia Francisellaspecies Francisella tularensis Fusobacterium mortiferum Fusobacteriumnecrogenes Fusobacterium necrophorum Fusobacterium nucleatumFusobacterium species Fusobacterium varium Gaffkya species Gardnerellavaginalis Gemella haemolysans Gemella morbillorum Gemella speciesGeotrichum candidum Geotrichum fermentans Geotrichum penicillarumGeotrichum penicillatum Geotrichum species Gordona species Haemophilusaegyptius Haemophilus aphrophilus Haemophilus ducreyi Haemophilushaemoglobinophilus Haemophilus haemolyticus Haemophilus influenzaeHaemophilus influenzae biotype I Haemophilus influenzae biotype IIHaemophilus influenzae biotype III Haemophilus influenzae biotype IVHaemophilus influenzae biotype V Haemophilus influenzae biotype VIHaemophilus influenzae biotype VII Haemophilus influenzae biotype VIIIHaemophilus paragallinarum Haemophilus parahaemolyticus Haemophilusparainfluenzae Haemophilus parainfluenzae biotype I Haemophilusparainfluenzae biotype II Haemophilus parainfluenzae biotype IIIHaemophilus parainfluenzae biotype IV Haemophilus parainfluenzae biotypeV Haemophilus parainfluenzae biotype VI Haemophilus parainfluenzaebiotype VII Haemophilus parainfluenzae biotype VIII Haemophilusparaphrohaemolyticus Haemophilus paraphrophilus Haemophilus segnisHaemophilus somnus Haemophilus species Hafnia alvei Hanseniasporaguilliermondii Hanseniaspora uvarum Hanseniaspora valbyensis Hansenulaanomala Hansenula holstii Hansenula polymorpha Helicobacter(Campylobacter) cinaedi Helicobacter (Campylobacter) fennelliaeHelicobacter (Campylobacter) pylori Issatchenkia orientalis Kingelladenitrificans Kingella indologenes Kingella kingae Kingella speciesKlebsiella ornithinolytica Klebsiella oxytoca Klebsiella planticolaKlebsiella pneumoniae subsp. ozaenae Klebsiella pneumoniae subsp.pneumoniae Klebsiella pneumoniae subsp. rhinoscleromatis Klebsiellaspecies Klebsiella terrigena Kloeckera apiculata Kloeckera apisKloeckera japonica Kloeckera species Kluyvera ascorbata Kluyveracryocrescens Kluyvera species Kluyveromyces lactis Kluyveromycesmarxianus Kluyveromyces thermotolerans Kocuria (Micrococcus) kristinaeKocuria (Micrococcus) rosea Kocuria(Micrococcus) varians Koserellatrabulsii Kytococcus (Micrococcus) sedentarius Lactobacillus (Weissella)viridescens Lactobacillus A Lactobacillus acidophilus Lactobacillus BLactobacillus brevis Lactobacillus buchneri Lactobacillus caseiLactobacillus casei subsp. casei Lactobacillus casei subsp. lactosusLactobacillus casei subsp. rhamnosus Lactobacillus catenaformisLactobacillus cellobiosus Lactobacillus collinoides Lactobacilluscoprophilus Lactobacillus crispatus Lactobacillus curvatus Lactobacillusdelbrueckii subsp. bulgaricus Lactobacillus delbrueckii subsp.delbrueckii Lactobacillus delbrueckii subsp. lactis Lactobacillusfermentum Lactobacillus fructivorans Lactobacillus helveticusLactobacillus helveticus subsp. jugurti Lactobacillus jenseniiLactobacillus lindneri Lactobacillus minutus Lactobacillus paracaseisubsp. paracasei Lactobacillus pentosus Lactobacillus plantarumLactobacillus salivarius Lactobacillus salivarius var. saliciniusLactobacillus species Lactococcus diacitilactis Lactococcus garvieaeLactococcus lactis subsp. cremoris Lactococcus lactis subsp.diacitilactis Lactococcus lactis subsp. hordniae Lactococcus lactissubsp. lactis Lactococcus plantarum Lactococcus raffinolactis Leclerciaadecarboxylata Legionella species Leminorella species Leptospira speciesLeptotrichia buccalis Leuconostoc (Weissella) paramesenteroidesLeuconostoc carnosum Leuconostoc citreum Leuconostoc gelidum Leuconostoclactis Leuconostoc mesenteroides Leuconostoc mesenteroides subsp.cremoris Leuconostoc mesenteroides subsp. dextranicum Leuconostocmesenteroides subsp. mesenteroides Leuconostoc species Listeria grayiListeria innocua Listeria ivanovii Listeria monocytogenes Listeriamurrayi Listeria seeligeri Listeria species Listeria welshimeriMegasphaera elsdenii Methylobacterium mesophilicum Metschnikowiapulcherrima Microbacterium species Micrococcus luteus Micrococcus lylaeMicrococcus species Mobiluncus curtisii Mobiluncus mulieris Mobiluncusspecies Moellerella wisconsensis Moraxella (Branhamella) catarrhalisMoraxella atlantae Moraxella bovis Moraxella lacunata Moraxellanonliquefaciens Moraxella osloensis Moraxella phenylpyruvica Moraxellaspecies Morganella morganii Morganella morganii subsp. morganiiMorganella morganii subsp. sibonii Mycobacterium africanum Mycobacteriumasiaticum Mycobacterium avium Mycobacterium bovis Mycobacterium chelonaeMycobacterium fortuitum Mycobacterium gordonae Mycobacterium kansasiiMycobacterium malmoense Mycobacterium marinum Mycobacterium phleiMycobacterium scrofulaceum Mycobacterium smegmatis Mycobacterium speciesMycobacterium tuberculosis Mycobacterium ulcerans Mycobacterium xenopiMycoplasma fermentans Mycoplasma hominis Mycoplasma orale Mycoplasmapneumoniae Mycoplasma species Myroides species Neisseria cinereaNeisseria elongata subsp. elongata Neisseria flava Neisseria flavescensNeisseria gonorrhoeae Neisseria lactamica Neisseria meningitidisNeisseria mucosa Neisseria perflava Neisseria polysaccharea Neisseriasaprophytes Neisseria sicca Neisseria subflava Neisseria weaveriNeisseria weaveri (CDC group M5) Nocardia species Ochrobactrum anthropiOerskovia species Oerskovia xanthineolytica Oligella (Moraxella)urethralis Oligella species Oligella ureolytica Paenibacillus alveiPaenibacillus macerans Paenibacillus polymyxa Pantoea agglomeransPantoea ananas (Erwinia uredovora) Pantoea dispersa Pantoea speciesPantoea stewartii Pasteurella (Haemophilus) avium Pasteurella aerogenesPasteurella gallinarum Pasteurella haemolytica Pasteurella haemolyticusPasteurella multocida Pasteurella multocida SF Pasteurella multocidasubsp. multocida Pasteurella multocida subsp. septica Pasteurellapneumotropica Pasteurella species Pasteurella ureae Pediococcusacidilactici Pediococcus damnosus Pediococcus pentosaceus Pediococcusspecies Peptococcus niger Peptococcus species Peptostreptococcusanaerobius Peptostreptococcus asaccharolyticus Peptostreptococcusindolicus Peptostreptococcus magnus Peptostreptococcus microsPeptostreptococcus parvulus Peptostreptococcus prevotiiPeptostreptococcus productus Peptostreptococcus speciesPeptostreptococcus tetradius Phaecoccomyces exophialiae Photobacteriumdamselae Pichia (Hansenula) anomala Pichia (Hansenula) jadinii Pichia(Hansenula) petersonii Pichia angusta (Hansenula polymorpha) Pichiacarsonii (P. vini) Pichia etchellsii Pichia farinosa Pichia fermentansPichia membranaefaciens Pichia norvegensis Pichia ohmeri Pichiaspartinae Pichia species Plesiomonas shigelloides Porphyromonasasaccharolytica Porphyromonas endodontalis Porphyromonas gingivalisPorphyromonas levii Prevotella (Bacteroides) buccae Prevotella(Bacteroides) buccalis Prevotella (Bacteroides) corporis Prevotella(Bacteroides) denticola Prevotella (Bacteroides) loescheii Prevotella(Bacteroides) oralis Prevotella (Bacteroides)disiens Prevotella(Bacteroides)oris Prevotella bivia (Bacteroides bivius) Prevotellaintermedia (Bacteroides intermedius) Prevotella melaninogenica(Bacteroides melaninogenicus) Prevotella ruminicola Propionibacteriumacnes Propionibacterium avidum Propionibacterium granulosumPropionibacterium propionicum Propionibacterium species Proteusmirabilis Proteus penneri Proteus species Proteus vulgaris Protothecaspecies Prototheca wickerhamii Prototheca zopfii Providenciaalcalifaciens Providencia heimbachae Providencia rettgeri Providenciarustigianii Providencia species Providencia stuartii Providenciastuartii urea + Pseudomonas (Chryseomonas) luteola Pseudomonasacidovorans Pseudomonas aeruginosa Pseudomonas alcaligenes Pseudomonascepacia Pseudomonas chlororaphis (P. aureofaciens) Pseudomonasfluorescens Pseudomonas fluorescens group Pseudomonas mendocinaPseudomonas pseudoalcaligenes Pseudomonas putida Pseudomonas speciesPseudomonas stutzeri Pseudomonas testosteroni Pseudomonas vesicularisPseudoramibacter (Eubacterium) alactolyticus Psychrobacter (Moraxella)phenylpyruvicus Rahnella aquatilis Ralstonia (Pseudomonas, Burkholderia)pickettii Rhodococcus (Corynebacterium) equi Rhodococcus speciesRhodosporidium toruloides Rhodotorula glutinis Rhodotorula minutaRhodotorula mucilaginosa (R. rubra) Rhodotorula species Rickettsiaspecies Rothia dentocariosa Saccharomyces cerevisiae Saccharomycesexiguus Saccharomyces kluyverii Saccharomyces species Sakaguchiadacryoides (Rhodosporidium dacryoidum) Salmonella arizonae Salmonellacholeraesuis Salmonella enteritidis Salmonella gallinarum Salmonellaparatyphi A Salmonella paratyphi B Salmonella pullorum Salmonellaspecies Salmonella typhi Salmonella typhimurium Salmonella typhisuisSalmonella/Arizona Serratia ficaria Serratia fonticola Serratia grimesiiSerratia liquefaciens Serratia marcescens Serratia odorifera Serratiaodorifera type 1 Serratia odorifera type 2 Serratia plymuthica Serratiaproteamaculans Serratia proteamaculans subsp. proteamaculans Serratiaproteamaculans subsp. quinovora Serratia rubidaea Serratia speciesShewanella (Pseudomonas, Alteromonas) putrefaciens Shigella boydiiShigella dysenteriae Shigella flexneri Shigella sonnei Shigella speciesSphingobacterium multivorum Sphingobacterium species Sphingobacteriumspiritivorum Sphingobacterium thalpophilum Sphingomonas (Pseudomonas)paucimobilis Sporidiobolus salmonicolor Sporobolomyces roseusSporobolomyces salmonicolor Sporobolomyces species Staphylococcus(Peptococcus) saccharolyticus Staphylococcus arlettae Staphylococcusaureus Staphylococcus aureus (Coagulase-negative) Staphylococcusauricularis Staphylococcus capitis Staphylococcus capitis subsp. capitisStaphylococcus capitis subsp. ureolyticus Staphylococcus capraeStaphylococcus carnosus Staphylococcus caseolyticus Staphylococcuschromogenes Staphylococcus cohnii Staphylococcus cohnii subsp. cohniiStaphylococcus cohnii subsp. urealyticum Staphylococcus epidermidisStaphylococcus equorum Staphylococcus gallinarum Staphylococcushaemolyticus Staphylococcus hominis Staphylococcus hominis subsp.hominis Staphylococcus hominis subsp. novobiosepticus Staphylococcushyicus Staphylococcus intermedius Staphylococcus kloosii Staphylococcuslentus Staphylococcus lugdunensis Staphylococcus saprophyticusStaphylococcus schleiferi Staphylococcus sciuri Staphylococcus simulansStaphylococcus species Staphylococcus warneri Staphylococcus xylosusStenotrophomonas (Xanthomonas) maltophilia Stephanoascus ciferriiStomatococcus mucilaginosus Streptococcus acidominimus Streptococcusagalactiae Streptococcus agalactiae (Group B) Streptococcus agalactiaehemolytic Streptococcus agalactiae non-hemolytic Streptococcusalactolyticus Streptococcus anginosus Streptococcus anginosus (Group D,nonenterococci) Streptococcus beta-hemolytic group A Streptococcusbeta-hemolytic non- group A or B Streptococcus beta-hemolytic non-groupA Streptococcus beta-hemolytic Streptococcus bovis (Group D,nonenterococci) Streptococcus bovis I Streptococcus bovis IIStreptococcus canis Streptococcus constellatus Streptococcusconstellatus (Streptococcus milleri I) Streptococcus constellatus(viridans Streptococcus) Streptococcus downei Streptococcus dysgalactiaesubsp. dysgalactiae Streptococcus dysgalactiae subsp. equisimilisStreptococcus equi (Group C/Group G Streptococcus) Streptococcus equisubsp. equi Streptococcus equi subsp. zooepidemicus Streptococcusequinus Streptococcus equinus (Group D, nonenterococci) Streptococcusequisimilis Streptococcus equisimulis (Group C/Group G Streptococcus)Streptococcus Gamma (non)-hemolytic Streptococcus gordonii StreptococcusGroup B Streptococcus Group C Streptococcus Group D Streptococcus GroupE Streptococcus Group F Streptococcus Group G Streptococcus Group LStreptococcus Group P Streptococcus Group U Streptococcus intermediusStreptococcus intermedius (Streptococcus milleri II) Streptococcusintermedius (viridans Streptococcus) Streptococcus milleri groupStreptococcus mitis Streptococcus mitis (viridans Streptococcus)Streptococcus mitis group Streptococcus mutans Streptococcus mutans(viridans Streptococcus) Streptococcus oralis Streptococcus parasanguisStreptococcus pneumoniae Streptococcus porcinus Streptococcus pyogenesStreptococcus pyogenes (Group A) Streptococcus salivarius Streptococcussalivarius (viridans Streptococcus) Streptococcus salivarius subsp.salivarius Streptococcus salivarius subsp. thermophilus Streptococcussanguis Streptococcus sanguis I (viridans Streptococcus) Streptococcussanguis II Streptococcus sanguis II (viridans Streptococcus)Streptococcus sobrinus Streptococcus species Streptococcus suis IStreptococcus suis II Streptococcus uberis Streptococcus uberis(viridans Streptococcus) Streptococcus vestibularis Streptococcuszooepidemicus Streptococcus zooepidemicus (Group C) Streptomycessomaliensis Streptomyces species Suttonella (Kingella) indologenesTatumella ptyseos Tetragenococcus (Pediococcus) halophilus Torulasporadelbrueckii (Saccharomyces rosei) Torulopsis candida Torulopsishaemulonii Torulopsis inconspicua Treponema species Trichosporon asahiiTrichosporon asteroides Trichosporon beigelii Trichosporon cutaneumTrichosporon inkin Trichosporon mucoides Trichosporon ovoidesTrichosporon pullulans Trichosporon species Turicella otitidisUreaplasma species Ureaplasma urealyticum Veillonella parvula (V.alcalescens) Veillonella species Vibrio alginolyticus Vibrio choleraeVibrio damsela Vibrio fluvialis Vibrio fumissii Vibrio harveyi Vibriohollisae Vibrio metschnikovii Vibrio mimicus Vibrio parahaemolyticusVibrio species Vibrio species SF Vibrio vulnificus Weeksella (Bergeylla)virosa Weeksella species Weeksella virosa Williopsis (Hansenula)saturnus Xanthomonas campestris Xanthomonas species Yarrowia (Candida)lipolytica Yersinia aldovae Yersinia enterocolitica Yersiniaenterocolitica group Yersinia frederiksenii Yersinia intermedia Yersiniaintermedius Yersinia kristensenii Yersinia pestis Yersiniapseudotuberculosis Yersinia pseudotuberculosis SF Yersinia ruckeriYersinia species Yokenella regensburgei Yokenella regensburgei(Koserella trabulsii) Zygoascus hellenicus Zygosaccharomyces species¹The list includes microorganisms that may be identified by APIidentification test systems and VITEK ® automated identification systemfrom bioMérieux Inc., or by the MicroScan ®-WalkAway ® automated systemsfrom Dade Behring. Identification relies on classical identificationmethods using batteries of biochemical and other phenotypical tests.

TABLE 16 tuf gene sequences obtained in our laboratory (Example 42).GenBank Species Strain no. Gene Accession no.* Abiotrophia adiacensATCC49175 tuf AF124224 Enterococcus avium ATCC14025 tufA AF124220 tufBAF274715 Enterococcus casseliflavus ATCC25788 tufA AF274716 tufBAF274717 Enterococcus cecorum ATCC43198 tuf AF274718 Enterococcuscolumbae ATCC51263 tuf AF274719 Enterococcus dispar ATCC51266 tufAAF274720 tufB AF274721 Enterococcus durans ATCC19432 tufA AF274722 tufBAF274723 Enterococcus faecalis ATCC29212 tuf AF124221 Enterococcusfaecium ATCC 19434 tufA AF124222 tufB AF274724 Enterococcus gallinarumATCC49573 tufA AF124223 tufB AF274725 Enterococcus hirae ATCC8043 tufAAF274726 tufB AF274727 Enterococcus malodoratus ATCC43197 tufA AF274728tufB AF274729 Enterococcus mundtii ATCC43186 tufA AF274730 tufB AF274731Enterococcus pseudoavium ATCC49372 tufA AF274732 tufB AF274733Enterococcus raffinosus ATCC49427 tufA AF274734 tufB AF274735Enterococcus saccharolyticus ATCC43076 tuf AF274736 Enterococcussolitarius ATCC49428 tuf AF274737 Enterococcus sulfureus ATCC49903 tufAF274738 Lactococcus lactis ATCC11154 tuf AF274745 Listeriamonocytogenes ATCC15313 tuf AF274746 Listeria seeligeri ATCC35967 tufAF274747 Staphylococcus aureus ATCC25923 tuf AF274739 Staphylococcusepidermidis ATCC14990 tuf AF274740 Streptococcus mutans ATCC25175 tufAF274741 Streptococcus pneumoniae ATCC6303 tuf AF274742 Streptococcuspyogenes ATCC19615 tuf AF274743 Streptococcus suis ATCC43765 tufAF274744 *Corresponding sequence ID NO. for the above ATCC strains aregiven in table 7.

TABLE 17 tuf gene sequences selected from databases for Example 42.Species Gene Accession no.* Agrobacterium tumefaciens tufA X99673 tufBX99674 Anacystis nidulans tuf X17442 Aquifex aeolicus tufA AE000657 tufBAE000657 Bacillus stearothermophilus tuf AJ000260 Bacillus subtilis tufAL009126 Bacteroides fragilis tuf P33165 Borrelia burgdorferi tufAE000783 Brevibacterium linens tuf X76863 Bulkholderia cepacia tufP33167 Campylobacter jejuni tufB Y17167 Chlamydia pneumoniae tufAE001363 Chlamydia trachomatis tuf M74221 Corynebacterium glutamicum tufX77034 Cytophaga lytica tuf X77035 Deinococcus radiodurans tuf AE000513Escherichia coli tufA J01690 tufB J01717 Fervidobacterium islandicum tufY15788 Haemophilus influenzae tufA L42023 tufB L42023 Helicobacterpylori tuf AE000511 Homo sapiens (Human) EF-1α X03558 Methanococcusjannaschii EF-1α U67486 Mycobacterium leprae tuf D13869 Mycobacteriumtuberculosis tuf X63539 Mycoplasma genitalium tuf L43967 Mycoplasmapneumoniae tuf U00089 Neisseria gonorrhoeae tufA L36380 Nicotianatabacum (Tobacco) EF-1α U04632 Peptococcus niger tuf X76869 Planobisporarosea tuf1 U67308 Saccharomyces cerevisiae (Yeast) EF-1α X00779Salmonella typhimurium tufA X55116 tufB X55117 Shewanella putrefacienstuf P33169 Spirochaeta aurantia tuf X76874 Spirulina platensis tufAX15646 Streptomyces aureofaciens tuf1 AF007125 Streptomyces cinnamoneustuf1 X98831 Streptomyces coelicolor tuf1 X77039 tuf3 X77040 Streptomycescollinus tuf1 S79408 Streptomyces ramocissimus tuf1 X67057 tuf2 X67058tuf3 X67059 Synechocystis sp. tuf AB001339 Taxeobacter ocellatus tufX77036 Thermotoga maritima tuf AE000512 Thermus aquaticus tuf X66322Thermus thermophilus tuf X06657 Thiobacillus cuprinus tuf U78300Treponema pallidum tuf AE000520 Wolinella succinogenes tuf X76872*Sequence data were obtained from GenBank, EMBL, and SWISSPROTdatabases. Genes were designated as appeared in the references.

Table 18 Nucleotide and amino acid sequence identities of EF-Tu betweendifferent enterococci and other low G + C gram-positive bacteria. Theupper right triangle represents the deduced amino acid sequenceidentities of gram-positive bacterial EF-Tu, while the lower lefttriangle represents the DNA sequence identities of the corresponding tufgenes. The sequence identities between different enterococcal tufA genesare boxed while those between enterococcal tufB genes are shaded.

TABLE 19 Strains analyzed in Example 43. 16S rDNA sequence accessionTaxon Strain* Strain† number Cedecea devisee ATCC 33431^(T) Cedecealapagei ATCC 33432^(T) Cedecea neteri ATCC 33855^(T) Citrobacteramalonaticus ATCC 25405^(T) CDC 9020-77^(T) AF025370 Citrobacter braakiiATCC 43162 CDC 080-58^(T) AF025368 Citrobacter farmeri ATCC 51112^(T)CDC 2991-81^(T) AF025371 Citrobacter freundii ATCC 8090^(T) DSM30039^(T) AJ233408 Citrobacter koseri ATCC 27156^(T) Citrobactersedlakii ATCC 51115^(T) CDC 4696-86^(T) AF025364 Citrobacter werkmaniiATCC 51114^(T) CDC 0876-58^(T) AF025373 Citrobacter youngae ATCC29935^(T) Edwardsiella hoshinae ATCC 33379^(T) Edwardsiella tarda ATCC15947^(T) CDC 4411-68 AF015259 Enterobacter aerogenes ATCC 13048^(T) JCM1235^(T) AB004750 Enterobacter agglomerans ATCC 27989 Enterobacteramnigenus ATCC 33072^(T) JCM 1237^(T) AB004749 Enterobacter asburiaeATCC 35953^(T) JCM 6051^(T) AB004744 Enterobacter cancerogenus ATCC35317^(T) Enterobacter cloacae ATCC 13047^(T) Enterobacter gergoviaeATCC 33028^(T) JCM 1234^(T) AB004748 Enterobacter hormaechei ATCC49162^(T) Enterobacter sakazakii ATCC 29544^(T) JCM 1233^(T) AB004746Escherichia coli ATCC 11775^(T) ATCC 11775^(T) X80725 Escherichia coliATCC 25922 ATCC 25922 X80724 Escherichia coli (ETEC) ATCC 35401Escherichia coli (O157:H7) ATCC 43895 ATCC 43895 Z83205 Escherichiafergusonii ATCC 35469^(T) Escherichia hermanii ATCC 33650^(T)Escherichia vulneris ATCC 33821^(T) ATCC 33821^(T) X80734 Ewingellaamericana ATCC 33852^(T) NCPPB 3905 X88848 Hafnia alvei ATCC 13337^(T)ATCC 13337^(T) M59155 Klebsiella omithinolytica ATCC 31898 CIP 103.364U78182 Klebsiella oxytoca ATCC 33496 ATCC 13182^(T) U78183 Klebsiellaplanticola ATCC 33531^(T) JCM 7251^(T) AB004755 Klebsiella pneumoniaesubsp. pneumoniae ATCC 13883^(T) DSM 30104^(T) AJ233420 subsp. ozaenaeATCC 11296^(T) ATCC 11296^(T) Y17654 subsp. rhinoscleromatis ATCC13884^(T) Kluyvera ascorbata ATCC 33433^(T) ATCC 14236 Y07650 Kluyveracryocrescens ATCC 33435^(T) Kluyvera georgiana ATCC 51603^(T) Leclerciaadecarboxylata ATCC 23216^(T) Leminorella grimontii ATCC 33999^(T) DSM5078^(T) AJ233421 Moellerella wisconsensis ATCC 35017^(T) Morganellamorganii ATCC 25830^(T) Pantoea agglomerans ATCC 27155^(T) DSM 3493^(T)AJ233423 Pantoea dispersa ATCC 14589^(T) Plesiomonas shigellokles ATCC14029^(T) Pragia fontium ATCC 49100^(T) DSM 5563^(T) AJ233424 Proteusmirabilis ATCC 25933 Proteus penneri ATCC 33519^(T) Proteus vulgarisATCC 13315^(T) DSM 30118^(T) AJ233425 Providencia alcalifaciens ATCC9886^(T) Providencia rettgeri ATCC 9250 Providencia rustigianii ATCC33673^(T) Providencia stuartii ATCC 33672 Rahnella aquatilis ATCC33071^(T) DSM 4594^(T) AJ233426 Salmonella choleraesuis subsp. arizonaeATCC 13314^(T) subsp. choleraesuis serotype Choleraesuis ATCC 7001serotype Enteritidis‡ ATCC 13076^(T) SE22 SE22 serotype Gallinarum ATCC9184 serotype Heidelberg ATCC 8326 serotype Paratyphi A ATCC 9150serotype Paratyphi B ATCC 8759 serotype Typhi‡ ATCC 10749 St111 U88545serotype Typhimurium‡ ATCC 14028 serotype Virchow ATCC 51955 subsp.diarizonae ATCC 43973^(T) subsp. houtenae ATCC 43974^(T) subsp. indicaATCC 43976^(T) subsp. salamae ATCC 43972^(T) Serratia fonticola DSM4576^(T) DSM 4576^(T) AJ233429 Serratia grimesii ATCC 14460^(T) DSM30063^(T) AJ233430 Serratia liquefaciens ATCC 27592^(T) Serratiamarcescens ATCC 13880^(T) DSM 30121^(T) AJ233431 Serratia odorifera ATCC33077^(T) DSM 4582^(T) AJ233432 Serratia plymuthica DSM 4540^(T) DSM4540^(T) AJ233433 Serratia rubidaea DSM 4480^(T) DSM 4480^(T) AJ233436Shigella boydii ATCC 9207 ATCC 9207 X96965 Shigella dysenteriae ATCC11835 ATCC 13313^(T) X96966 ATCC 25931 X96964 Shigella flexneri ATCC12022 ATCC 12022 X96963 Shigella sonnei ATCC 29930^(T) Tatumella ptyseosATCC 33301^(T) DSM 50001 AJ233437 Trabulsiella guamensis ATCC 49490^(T)Yersinia enterocolitica ATCC 9610^(T) ATCC 9610^(T) M59292 Yersiniafrederiksenii ATCC 33641^(T) Yersinia intermedia ATCC 29909^(T) Yersiniapestis RRB KIMD27 ATCC 19428^(T) X75274 Yersinia pseudotuberculosis ATCC29833^(T) Yersinia rohdei ATCC 43380^(T) ER-2935^(T) X75276 Shewanellaputrefaciens ATCC 8071^(T) Vibrio cholerae ATCC 25870 ATCC 14035^(T)X74695 ^(T)Type strain *Strains used in this study for sequencing ofpartial tuf and atpD genes. SEQ ID NOs. for tuf and atpD sequencescorresponding to the above reference strains are given in table 7.†Strains used in other studies for sequencing of 16S rDNA gene. Whenboth strain numbers are on the same row, both strains are considered tobe the same although strain numbers may be different. ‡Phylogeneticserotypes considered species by the Bacteriological Code (1990Revision).

TABLE 20 PCR primer pairs used in this study Primer Nucleotide AmpliconSEQ ID NO. Sequence positions* length (bp) tuf 6645′-AAYATGATIACIGGIGCIGCICARATGGA-3′ 271-299 884 6975′-CCIACIGTICKICCRCCYTCRCG-3′ 1132-1156 atpD 5685′-RTIATIGGIGCIGTIRTIGAYGT-3′ 25-47 884 5675′-TCRTCIGCIGGIACRTAIAYIGCYTG-3′ 883-908 7005′-TIRTIGAYGTCGARTTCCCTCARG-3′ 38-61 871 5675′-TCRTCIGCIGGIACRTAIAYIGCYTG-3′ 883-908 *The nucleotide positions givenare for E. coli tuf and atpD sequences (GenBank accession no. AE000410and V00267, respectively). Numbering starts from the first base of theinitiation codon.

TABLE 21Selection of M. catarrhalis-specific primer pairs from SEQ ID NO: 29¹ (466 bp DNA fragment) other than those previously tested².Amplicon Moraxella Moraxella Moraxella Moraxella size catarrhaliscatarrhalis non- Moraxella Moraxella Moraxella phenyl- Primer Sequence(bp) ATCC 43628 ATCC 53879 liquefaciens lacunata osloensis atlantaepyruvica SEQ ID NO: 118 CGCTGACGGCTTGTTTGTACCA 118 +³ + − − − − −SEQ ID NO: 119 TGTTTTGAGCTTTTTATTTTTTGA VBmcat1TGCTTAAGATTCACTCTGCCATTTT  93 + + − − − − − (SEQ ID NO: 2298) VBmcat2TAAGTCGCTGACGGCTTGTTT (SEQ ID NO: 2299) VBmcat3 CCTGCACCACAAGTCATCAT140 + + − − − − − (SEQ ID NO: 2300) VBmcat4 AATTCACCAACAATGTCAAAGC(SEQ ID NO: 2301) VBmcat5 AATGATAACCAGTCAAGCAAGC 219 + + − − − − −(SEQ ID NO: 2302) VBmcat6 GGTGCATGGTGATTTGTAAAA (SEQ ID NO: 2303)VBmcat7 GTGTGCGTTCACTTTTACAAAT 160 + + − − − − − (SEQ ID NO: 2304)VBmcat8 GGTGTTAAGCTGATGATGAGAG (SEQ ID NO: 2305) VBmcat9TGACCATGCACACCCTTATT 167 + + − − − − − (SEQ ID NO: 2306) VBmcat10TCATTGGGATGAAAGTATCGTT (SEQ ID NO: 2307) Amplicon size Kingella KingellaNeisseria Neisseria Escherichia Staphylococcus Primer Sequence (bp)indologenes kingea meningitidis gonorrhoeae coli aureus SEQ ID NO: 118CGCTGACGGCTTGTTTGTACCA 118 − − − − − − SEQ ID NO: 119TGTTTTGAGCTTTTTATTTTTTGA VBmcat1 TGCTTAAGATTCACTCTGCCATTTT  93 − − − − −− (SEQ ID NO: 2298) VBmcat2 TAAGTCGCTGACGGCTTGTTT (SEQ ID NO: 2299)VBmcat3 CCTGCACCACAAGTCATCAT 140 − − − − − − (SEQ ID NO: 2300) VBmcat4AATTCACCAACAATGTCAAAGC (SEQ ID NO: 2301) VBmcat5 AATGATAACCAGTCAAGCAAGC219 − − − − − − (SEQ ID NO: 2302) VBmcat6 GGTGCATGGTGATTTGTAAAA(SEQ ID NO: 2303) VBmcat7 GTGTGCGTTCACTTTTACAAAT 160 − − − − − −(SEQ ID NO: 2304) VBmcat8 GGTGTTAAGCTGATGATGAGAG (SEQ ID NO: 2305)VBmcat9 TGACCATGCACACCCTTATT 167 − − − − − − (SEQ ID NO: 2306) VBmcat10TCATTGGGATGAAAGTATCGTT (SEQ ID NO: 2307) ¹SEQ ID NO. from U.S. Pat. No.6,001,564. ²All PCR assays were performed with 1 ng of purified genomicDNA by using an annealing temperature of 55° C. and 30 cycles ofamplification. The genomic DNA from the various bacterial species abovewas always isolated from reference strains obtained from ATCC. ³Allpositive results showed a strong amplification signal with genomic DNAfrom the target species M. catarrhalis.

TABLE 22Selection of S. epidermidis-specific primer pairs from SEQ ID NO: 36¹ (705 bp DNA fragment) other than those previously tested. Amplicon Staphylococcus Staphylococcus Staphylo-Staphylo- Staphylo- Staphylo- Sequence size epidermidis epidermidiscoccus coccus coccus coccus Staphylo- Primer (all 25 nucleotides) (bp)ATCC 14990 ATCC 12228 capitis cohnii aureus auricularis coccusSEQ ID NO: 145 ATCAAAAAGTTGGCGAACCTTTTCA 125 +³ + − − − − −SEQ ID NO: 146 CAAAAGAGCGTGGAGAAAAGTATCA VBsep3 (SEQ ID NO: 2308)CATAGTCTGATTGCTCAAAGTCTTG 208 + + − − − − + VBsep4 (SEQ ID NO: 2309)GCGAATAGTGAACTACATTCTGTTG + + − − − − − VBsep5 (SEQ ID NO: 2310)CACGCTCTTTTGCAATTTCCATTGA 208 + + + + + − + VBsep6 (SEQ ID NO: 2311)GAAGCAAATATTCAAAATGCACCAG + + + + + − + VBsep7 (SEQ ID NO: 2312)AAAGTCTTTTGCTTCTTCAGATTCA 177 + + − − − − + VBsep8 (SEQ ID NO: 2313)GTGTTCACAGGTATGGATGCTCTTA + + NT NT − NT − + + NT NT − NT −VBsep9 (SEQ ID NO: 2314) GAGCATCCATACCTGTGAACACAGA 153 + + − − − − +VBsep10 (SEQ ID NO: 2315) TTTTCCAATTACAAGAGACATCAGT + + NT NT − NT + + +NT NT − NT − VBsep11 (SEQ ID NO: 2316) TTTGAATTCGCATGTACTTTGTTTG 135 + +− − − − − VBsep12 (SEQ ID NO: 2317) CCCCGGGTTCGAAATCGATAAAAAG AmpliconStaphylo- Staphylo- Entero- Sequence size Staphylococcus coccus coccusBacillus coccus Primer (all 25 nucleotides) (bp) hominis StaphylococcusStaphylococcus simulans warneri subtilis faecalis SEQ ID NO: 145ATCAAAAAGTTGGCGAACCTTTTCA 125 − − − − − − − SEQ ID NO: 146CAAAAGAGCGTGGAGAAAAGTATCA VBsep3 (SEQ ID NO: 2308)CATAGTCTGATTGCTCAAAGTCTTG 208 − − − − − − − VBsep4 (SEQ ID NO: 2309)GCGAATAGTGAACTACATTCTGTTG − − − − − − − VBsep5 (SEQ ID NO: 2310)CACGCTCTTTTGCAATTTCCATTGA 208 + − − − − − − VBsep6 (SEQ ID NO: 2311)GAAGCAAATATTCAAAATGCACCAG + − − − NT NT NT VBsep7 (SEQ ID NO: 2312)AAAGTCTTTTGCTTCTTCAGATTCA 177 − − − + − − − VBsep8 (SEQ ID NO: 2313)GTGTTCACAGGTATGGATGCTCTTA NT − − + NT NT NT NT − − − NT NT NTVBsep9 (SEQ ID NO: 2314) GAGCATCCATACCTGTGAACACAGA 153 − + + − − − −VBsep10 (SEQ ID NO: 2315) TTTTCCAATTACAAGAGACATCAGT NT + − − NT NT NT NT− − − NT NT NT VBsep11 (SEQ ID NO: 2316) TTTGAATTCGCATGTACTTTGTTTG 135 −− − − − − − VBsep12 (SEQ ID NO: 2317) CCCCGGGTTCGAAATCGATAAAAAG AmpliconListeria Strepto- Strepto- Annealing Sequence size EnterococcusEnterococcus mono- coccus Streptococcus coccus temperature² Primer(all 25 nucleotides) (bp) faecium gallinarum cytogenes agalactieapneumoniae pyogenes (° C.) SEQ ID NO: 145 ATCAAAAAGTTGGCGAACCTTTTCA 125− − − − − − 55 SEQ ID NO: 146 CAAAAGAGCGTGGAGAAAAGTATCAVBsep3 (SEQ ID NO: 2308)  CATAGTCTGATTGCTCAAAGTCTTG 208 − − − − − − 55VBsep4 (SEQ ID NO: 2309) GCGAATAGTGAACTACATTCTGTTG − − − − − − 60VBsep5 (SEQ ID NO: 2310) CACGCTCTTTTGCAATTTCCATTGA 208 − − − − − − 55VBsep6 (SEQ ID NO: 2311) GAAGCAAATATTCAAAATGCACCAG NT NT NT NT NT NT 65VBsep7 (SEQ ID NO: 2312) AAAGTCTTTTGCTTCTTCAGATTCA 177 − − − − − − 55VBsep8 (SEQ ID NO: 2313) GTGTTCACAGGTATGGATGCTCTTA NT NT NT NT NT NT 60NT NT NT NT NT NT 65 VBsep9 (SEQ ID NO: 2314) GAGCATCCATACCTGTGAACACAGA153 − − − − − − 55 VBsep10 (SEQ ID NO: 2315) TTTTCCAATTACAAGAGACATCAGTNT NT NT NT NT NT 60 NT NT NT NT NT NT 65 VBsep11 (SEQ ID NO: 2316)TTTGAATTCGCATGTACTTTGTTTG 135 − − − − − − 55 VBsep12 (SEQ ID NO: 2317)CCCCGGGTTCGAAATCGATAAAAAG ¹SEQ ID NO. from U.S. Pat. No. 6,001,564. ²AllPCR assays were performed with 1 ng of purified genomic DNA by using anannealing temperature of 55 tp 65° C. and 30 cycles of amplification.The genomic DNA from the various bacterial species above was alwaysisolated from reference strains obtained from ATCC. ³All positiveresults showed a strong amplification signal with genomic DNA from thetarget species S. epidermidis. The instensity of the positiveamplification signal with species other than S. epidermidis wasvariable. NT = not tested.

TABLE 23Influence of nucleotide variation(s) on the efficiency of the PCR amplification:Example with SEQ ID NO: 146 from S.epidermidis.

¹All PCR tests were performed with SEQ ID NO: 145 without modificationcombined with SEQ ID NO: 146 or 13 modified versions of SEQ ID NO: 146.Boxed nucleotides indicate changes in SEQ ID NO: 146. ²The tests with S.epidermidis were performed by using an annealing temperature of 55°C.with 1, 0, 1 and 0,01 ng of purified genomic DNA or at 50°C. with 1 ngof purified genomic DNA. ³The tests with S. aureus were performed onlyat 50°C. with 1 ng of genomic DNA. ⁴The intensity of the positiveamplification signal was quantified as follows: 3+ = strong signal, 2+ =intermediate signal and += weak signal.

TABLE 24Effect of the primer length on the efficiency of the PCR amplification¹: Example with AT-rich SEQ ID NO: 145²and SEQ ID NO: 146² from S. epidermidis. II. STAPHYLOCOCCUS EPIDERMIDIS³ Staphylo- Staphylo- Staphylo- Staphylo- ATCC 14990 coccus coccuscoccus coccus Length 45° C. 55° C. aureus ⁴ haemolyticus capitis warneriPrimer Sequence (nt) 1 0.1 0.01 1 0.1 0.01 45 55 45 55 45 55 45 55VBsep301 (SEQ ID NO: 2331) ATATCATCAAAAAGTTGGCGAACCTTT 30 NT NT NT 4+ 3+2+ NT − NT − NT − NT − TCA VBsep302 (SEQ ID NO: 2332)AATTGCAAAAGAGCGTGGAGAAAAGTA 30 TCA SEQ ID NO: 145 ATCAAAAAGTTGGCGAACCTTTTCA 25  4+⁵ 3+ 2+ 4+ 3+ 2+ − − − − + − − −(SEQ ID NO: 2333) SEQ ID NO: 146  CAAAAGAGCGTGGAGAAAAGTATCA 25(SEQ ID NO: 2334) VBsep201 (SEQ ID NO: 2335) AAAGTTGGCGAACCTTTTCA 20 NTNT NT 4+ 3+ 2+ NT − NT − NT − NT − VBsep202 (SEQ ID NO: 2336)GAGCGTGGAGAAAAGTATCA 20 VBsep171 (SEQ ID NO: 2337) GTTGGCGAACCTTTTCA 174+ 3+ 2+ 3+ 2+ + − − − − − − − − VBsep172 (SEQ ID NO: 2338)CGTGGAGAAAAGTATCA 17 VBsep151 (SEQ ID NO: 2339) TGGCGAACCTTTTCA 15 3+2+ + − − − − − − − − − − − VBsep152 (SEQ ID NO: 2340) TGGAGAAAAGTATCA 15¹All PCR tests were performed using an annealing temperature of 45 or55° C. and 30 cycles of amplification. ²All SEQ ID NOs. in this Tableare from U.S. Pat. No. 6,001,546. ³The tests with S. epidermidis weremade with 1, 0.1 and 0.01 ng of purified genomic DNA. ⁴The tests withall other bacterial species were made only with 1 ng of purified genomicDNA. ⁵The intensity of the positive amplification signal was quantifiedas follows: 4+ = very strong signal, 3+ = strong signal, 2+ =intermediate signal and + = weak signal. NT = not tested.

TABLE 25Effect of the primer length on the efficiency of the PCR amplification¹: Example with the GC-rich SEQ ID NO: 83² and SEQ ID NO: 84² from P. aeruginosa. III.Steno- Haemo- PSEUDOMONA Pseudo- tro- Neis- philus S AERUGINOSA ³ monasBurk- Shewa- phomonas seria para- Length ATCC 35554 fluor- holderianella malto- menin- haemo- Primer Sequence (nt) 1 0.1 0.01 escens ⁴cepacia putida philia gitidis lyticus SEQ ID NO 83 CGAGCGGGTGGTGTTCATC19  2+⁵ + − − − − − − − SEQ ID NO 84 CAAGTCGTCGTCGGAGGGA 19 Pse554-16aCGAGCGGGTGGTGTTC 16 2+ + − − − − − − − (SEQ ID NO: 2341) Pse674-16aGTCGTCGTCGGAGGGA 16 (SEQ ID NO: 2342) Pse554-13b GCGGGTGGTGTTC 13 2+ + −− − − − − − (SEQ ID NO: 2343) Pse674-13a GTCGTCGGAGGGA 13 (SEQ ID NO:2344) ¹All PCR tests were performed using an annealing temperature of55° C. and 30 cycles of amplification. ²All SEQ ID NOs. in this Tableare from U.S. Pat. No. 6,001,546. ³The tests with P. aeruginosa weremade with 1, 0.1 and 0.01 ng of purified genomic DNA. ⁴The tests withall other bacterial species were made only with 1 ng of purified genomicDNA. ⁵The intensity of the positive amplification signal was quantifiedas follows: 2+ = strong signal and + = moderately strong signal.

TABLE 39Specific and ubiquitous primers for nucleic acid amplification (tuf sequences).Originating DNA fragment SEQ ID Nucleotide SEQ ID NO.Nucleotide sequence NO. positionBacterial species: Acinetobacter baumannii 16925′-GGT GAG AAC TGT GGT ATC TTA CTT   1 478-501 1693^(a)5′-CAT TTC AAC GCC TTC TTT CAA CTG   1 691-714Bacterial species: Chlamydia pneumoniae  6305′-CGG AGC TAT CCT AGT CGT TTC A  20   2-23  629^(a)5′-AAG TTC CAT CTC AAC AAG GTC AAT A  20 146-170 20855′-CAA ACT AAA GAA CAT ATC TTG CTA  20  45-68 2086^(a)5′-ATA TAA TTT GCA TCA CCT TCA AG  20 237-259 20875′-TCA GCT CGT GGG ATT AGG AGA G  20 431-452 2088^(a)5′-AGG CTT CAC GCT GTT AGG CTG A  20 584-605Bacterial species: Chlamydia trachomatis  5545′-GTT CCT TAC ATC GTT GTT TTT CTC  22  82-105  555^(a)5′-TCT CGA ACT TTC TCT ATG TAT GCA  22 249-272Parasitical species: Cryptosporidium parvum  7985′-TGG TTG TCC CAG CCG ATC GTT T 865 158-179  804^(a)5′-CCT GGG ACG GCC TCT GGC AT 865 664-683  7995′-ACC TGT GAA TAC AAG CAA TCT 865 280-300  805^(a)5′-CTC TTG TCC ATC TTA GCA GT 865 895-914  8005′-GAT GAA ATC TTC AAC GAA GTT GAT 865 307-330  806^(a)5′-AGC ATC ACC AGA CTT GAT AAG 865 946-966  8015′-ACA ACA CCG AGA AGA TCC CA 865 353-372  803^(a)5′-ACT TCA GTG GTA ACA CCA GC 865 616-635  8025′-TTG CCA TTT CTG GTT TCG TT 865 377-396  807^(a)5′-AAA GTG GCT TCA AAG GTT GC 865 981-1000Bacterial species: Enterococcus faecium 16965′-ATG TTC CTG TAG TTG CTG GA  64 189-208 1697^(a)5′-TTT CTT CAG CAA TAC CAA CAA C  64 422-443Bacterial species: Klebsiella pneumoniae 13295′-TGT AGA GCG CGG TAT CAT CAA AGT A 103 352-377 1330^(a)5′-AGA TTC GAA CTT GGT GTG CGG G 103 559-571^(a)These sequences are from the complementary DNA strand of the sequence of the originatingfragment given in the Sequence Listing. Originating DNA fragment SEQ IDNucleotide SEQ ID NO. Nucleotide sequence NO. positionBacterial species: Mycoplasma pneumoniae 20935′-TGT TGG CAA TCG AAG ACA CC  2097^(a) 635-654 2094^(b)5′-TTC AAT TTC TTG ACC TAC TTT CAA  2097^(a) 709-732Bacterial species: Neisseria gonorrhoeae  5515′-GAA GAA AAA ATC TTC GAA CTG GCT A  126 256-280  552^(b)5′-TAC ACG GCC GGT GAC TAC G  126 378-396 21735′-AAG AAA AAA TCT TCG AAC TGG CTA  126 257-280 2174^(b)5′-TCT ACA CGG CCG GTG  126 384-398 2175 5′-CCG CCA TAC CCC GTT T  126654-669 2176^(b) 5′-CGG CAT TAC CAT TTC CAC ACC TTT  126 736-759Bacterial species: Pseudomonas aeruginosa 16945′-AAG GCA AGG ATG ACA ACG GC  153 231-250 1695^(b)5′-ACG ATT TCC ACT TCT TCC TGG  153 418-438Bacterial species: Streptococcus agalactiae  5495′-GAA CGT GAT ACT GAC AAA CCT TTA 207-210^(c) 308-331^(d)  550^(b)5′-GAA GAA GAA CAC CAA CGT TG 207-210^(c) 520-539^(d)Bacterial species: Streptococcus pyogenes  9995′-TTG ACC TTG TTG ATG ACG AAG AG 1002 143-165 1000^(b)5′-TTA GTG TGT GGG TTG ATT GAA CT 1002 622-644 10015′-AAG AGT TGC TTG AAT TAG TTG AG 1002 161-183 1000^(b)5′-TTA GTG TGT GGG TTG ATT GAA CT 1002 622-644Parasitical species: Trypanosoma brucei  8205′-GAA GGA GGT GTC TGC TTA CAC  864 513-533  821^(b)5′-GGC GCA AAC GTC ACC ACA TCA  864 789-809  8205′-GAA GGA GGT GTC TGC TTA CAC  864 513-533  822^(b)5′-CGG CGG ATG TCC TTA ACA GAA  864 909-929 ^(a)Sequence from databases.^(b)These sequences are from the complementary DNA strand of the sequence of the originatingfragment given in the Sequence Listing.^(c)These sequences were aligned to derive the corresponding primer.^(d)The nucleotide positions refer to the S. agalactiae tuf sequence fragment(SEQ ID NO. 209). Originating DNA fragment SEQ ID Nucleotide SEQ ID NO.Nucleotide sequence NO. position Parasitical species: Trypanosoma cruzi 794 5′-GAC GAC AAG TCG GTG AAC TT 840-842^(a) 281-300^(c)  795^(b)5′-ACT TGC ACG CGA TGT GGC AG 840-842^(a) 874-893^(c)Bacterial genus: Clostridium sp.  796 5′-GGT CCA ATG CCW CAA ACW AGA32, 719-  32-52^(d) 724, 736^(a)  797^(b)5′-CAT TAA GAA TGG YTT ATC TGT SKC TCT 32, 719- 320-346^(d) 724, 736^(a) 808 5′-GCI TTA IWR GCA TTA GAA RAY CCA 32, 719- 224-247^(d)724, 736^(a)  809^(b) 5′-TCT TCC TGT WGC AAC TGT TCC TCT 32, 719-337-360^(d) 724, 736^(a)  810 5′-AGA GMW ACA GAT AAR SCA TTC TTA32, 719- 320-343^(d) 724, 736^(a)  811^(b)5′-TRA ART AGA ATT GTG GTC TRT ATC C 32, 719- 686-710^(d) 724, 736^(a)Bacterial genus: Corynebacterium sp.  5455′-TAC ATC CTB GTY GCI CTI AAC AAG TG 34-44, 662^(a)  89-114^(e) 546^(b) 5′-CCR CGI CCG GTR ATG GTG AAG AT 34-44, 662^(a) 350-372^(e)Bacterial genus: Enterococcus sp.  656 5′-AAT TAA TGG CTG CAG TTG AYG A58-72^(a) 273-294^(f)  657^(b) 5′-TTG TCC ACG TTC GAT RTC TTC A58-72^(a) 556-577^(f)  656 5′-AAT TAA TGG CTG CAG TTG AYG A 58-72^(a)273-294^(f)  271^(b) 5′-TTG TCC ACG TTG GAT RTC TTC A 58-72^(a)556-577^(f) 1137 5′-AAT TAA TGG CTG CWG TTG AYG AA 58-72^(a) 273-295^(f)1136^(b) 5′-ACT TGT CCA CGT TSG ATR TCT 58-72^(a) 559-579^(f)^(a)These sequences were aligned to derive the corresponding primer.^(b)These sequences are from the complementary DNA strand of the sequence of the originatingfragment given in the Sequence Listing.^(c)The nucleotide positions refer to the T. cruzi tuf sequence fragment (SEQ ID NO. 842).^(d)The nucleotide positions refer to the C. perfringens tuf sequence fragment(SEQ ID NO. 32).^(e)The nucleotide positions refer to the C. diphtheriae tuf sequence fragment(SEQ ID NO. 662).^(f)The nucleotide positions refer to the E. durans tuf sequence fragment (SEQ ID NO. 61).Originating DNA fragment SEQ ID Nucleotide SEQ ID NO.Nucleotide sequence NO. position Bacterial genus: Legionella sp. 20815′-GRA TYR TYA AAG TTG GTG AGG AAG 111-112^(a) 411-434^(b) 2082^(c)5′-CMA CTT CAT CYC GCT TCG TAC C 111-112^(a) 548-569^(b)Bacterial genus: Staphylococcus sp.  5535′-GGC CGT GTT GAA CGT GGT CAA ATC A 176-203^(a) 313-337^(d)  575^(c)5′-TIA CCA TTT CAG TAC CTT CTG GTA A 176-203^(a) 653-677^(d)  5535′-GGC CGT GTT GAA CGT GGT CAA ATC A 176-203^(a) 313-337^(d)  707^(c)5′-TWA CCA TTT CAG TAC CTT CTG GTA A 176-203^(a) 653-677^(d)Bacterial genus: Streptococcus sp.  5475′-GTA CAG TTG CTT CAG GAC GTA TC 206-231^(a) 372-394^(e)  548^(c)5′-ACG TTC GAT TTC ATC ACG TTG 206-231^(a) 548-568^(e)Fungal genus: Candida sp.  576 5′-AAC TTC RTC AAG AAG GTY GGT TAC AA407-426, 332-357^(f) 428-432^(a)  632^(c)5′-CCC TTT GGT GGR TCS TKC TTG GA 407-426, 791-813^(f) 428-432^(a)  6315′-CAG ACC AAC YGA IAA RCC ATT RAG AT 407-426, 523-548^(f) 428-432^(a) 632^(c) 5′-CCC TTT GGT GGR TCS TKC TTG GA 407-426, 791-813^(f)428-432^(a)  633 5′-CAG ACC AAC YGA IAA RCC ITT RAG AT 407-426,523-548^(f) 428-432^(a)  632^(c) 5′-CCC TTT GGT GGR TCS TKC TTG GA407-426, 791-813^(f) 428-432^(a)^(a)These sequences were aligned to derive the corresponding primer.^(b)The nucleotide positions refer to the L. pneumophila tuf sequence fragment(SEQ ID NO. 112).^(c)These sequences are from the complementary DNA strand of the sequence of the originatingfragment given in the Sequence Listing.^(d)The nucleotide positions refer to the S. aureus tuf sequence fragment (SEQ ID NO. 179).^(e)The nucleotide positions refer to the S. agalactiae tuf sequence fragment(SEQ ID NO. 209).^(f)The nucleotide positions refer to the C. albicans tuf(EF-1) sequence fragment(SEQ ID NO. 408). Originating DNA fragment SEQ ID Nucleotide SEQ ID NO.Nucleotide sequence NO. position Fungal genus: Cryptococcus sp. 19715′-CYG ACT GYG CCA TCC TYA TCA 434, 623, 1281,  150-170^(b)1985, 1986^(a) 1973^(c) 5′-RAC ACC RGI YTT GGW ITC CTT 434, 623, 1281, 464-484^(b) 1985, 1986^(a) 1972 5′-MGI CAG CTC ATY ITT GCW KSC434, 623, 1281,  260-280^(b) 1985, 1986^(a) 1973^(c)5′-RAC ACC RGI YTT GGW ITC CTT 434, 623, 1281,  464-484^(b)1985, 1986^(a) Parasitical genus: Entamoeba sp.  7035′-TAT GGA AAT TCG AAA CAT CT 512  38-57  704^(c)5′-AGT GCT CCA ATT AAT GTT GG 512 442-461  7035′-TAT GGA AAT TCG AAA CAT CT 512  38-57  705^(c)5′-GTA CAG TTC CAA TAC CTG AA 512 534-553  7035′-TAT GGA AAT TCG AAA CAT CT 512  38-57  706^(c)5′-TGA AAT CTT CAC ATC CAA CA 512 768-787  7935′-TTA TTG TTG CTG CTG GTA CT 512 149-168  704^(c)5′-AGT GCT CCA ATT AAT GTT GG 512 442-461 Parasitical genus: Giardia sp. 816 5′-GCT ACG ACG AGA TCA AGG GC 513 305-324  819^(c)5′-TCG AGC TTC TGG AGG AAG AG 513 895-914  8175′-TGG AAG AAG GCC GAG GAG TT 513 355-374  818^(c)5′-AGC CGG GCT GGA TCT TCT TC 513 825-844Parasitical genus: Leishmania sp.  701 5′-GTG TTC ACG ATC ATC GAT GCG514-526^(a)  94-114^(d)  702^(c) 5′-CTC TCG ATA TCC GCG AAG CG514-526^(a) 913-932^(d)^(a)These sequences were aligned to derive the corresponding primer.^(b)The nucleotide positions refer to the C. neoformans tuf (EF-1) sequence fragment(SEQ ID NO. 623).^(c)These sequences are from the complementary DNA strand of the sequence of the originatingfragment given in the Sequence Listing.^(d)The nucleotide positions refer to the L. tropica tuf(EF-1) sequence fragment(SEQ ID NO. 526). Originating DNA fragment SEQ ID Nucleotide SEQ ID NO.Nucleotide sequence NO. position Parasitical genus: Trypanosome sp.  8235′-GAG CGG TAT GAY GAG ATT GT 529, 840-  493-512^(b) 842, 864^(a) 824^(c) 5′-GGC TTC TGC GGC ACC ATG CG 529, 840- 1171-1190^(b)842, 864^(a) Bacterial family: Enterobacteriaceae  9335′-CAT CAT CGT ITT CMT GAA CAA RTG 78, 103, 146,  390-413^(d)168, 238, 698^(a)  934^(c) 5′-TCA CGY TTR RTA CCA CGC AGI AGA78, 103, 146,  831-854^(d) 168, 238, 698^(a)Bacterial family: Mycobacteriaceae  5395′-CCI TAC ATC CTB GTY GCI CTI AAC AAG  122   85-111  540^(c)5′-GGD GCI TCY TCR TCG WAI TCC TG  122  181-203Bacterial group: Escherichia coli and Shigella 16615′-TGG GAA GCG AAA ATC CTG 1668^(e)  283-300 1665^(c)5′-CAG TAC AGG TAG ACT TCT G 1668^(e)  484-502Bacterial group: Pseudomonads group  5415′-GTK GAA ATG TTC CGC AAG CTG CT 153-155^(a)  476-498^(f)  542^(c)5′-CGG AAR TAG AAC TGS GGA CGG TAG 153-155^(a)  679-702^(f)  5415′-GTK GAA ATG TTC CGC AAG CTG CT 153-155^(a)  476-498^(f)  544^(c)5′-AYG TTG TCG CCM GGC ATT MCC AT 153-155^(a)  749-771^(f)^(a)These sequences were aligned to derive the corresponding primer.^(b)The nucleotide positions refer to the T. brucei tuf (EF-1) sequence fragment(SEQ ID NO. 864).^(c)These sequences are from the complementary DNA strand of the sequence of theoriginating fragment given in the Sequence Listing.^(d)The nucleotide positions refer to the E. coli tuf sequence fragment(SEQ ID NO. 698). ^(e)Sequence from databases.^(f)The nucleotide positions refer to the P. aeruginosa tuf sequence fragment(SEQ ID NO. 153). Originating DNA fragment SEQ ID Nucleotide SEQ ID NO.Nucleotide sequence NO. positionParasitical group: Trypanosomatidae family 9235′-GAC GCI GCC ATC CTG ATG ATC 511, 514-526,  166-188^(b) 529, 840-842,864^(a) 924^(c) 5′-ACC TCA GTC GTC ACG TTG GCG 511, 514-526, 648-668^(b) 529, 840-842, 864^(a) 925 5′-AAG CAG ATG GTT GTG TGC TG511, 514-526,  274-293^(b) 529, 840-842, 864^(a) 926^(c)5′-CAG CTG CTC GTG GTG CAT CTC GAT 511, 514-526,  676-699^(b)529, 840-842, 864^(a) 927 5′-ACG CGG AGA AGG TGC GCT T 511, 514-526, 389-407^(b) 529, 840-842, 864^(a) 928^(c)5′-GGT CGT TCT TCG AGT CAC CGC A 511, 514-526,  778-799^(b)529, 840-842, 864^(a) Universal primers (bacteria) 6365′-ACT GGY GTT GAI ATG TTC CGY AA 7, 54, 78, 470-492^(d) 100, 103, 159,209, 224, 227^(b) 637^(c) 5′-ACG TCA GTI GTA CGG AAR TAG AA 7, 54, 78,692-714^(d) 100, 103, 159, 209, 224, 227^(b) 6385′-CCA ATG CCA CAA ACI CGT GAR CAC AT 7, 54, 78,  35-60^(e)100, 103, 159, 209, 224, 227^(b) 639^(c)5′-TTT ACG GAA CAT TTC WAC ACC WGT IAC A 7, 54, 78, 469-496^(e)100, 103, 159, 209, 224, 227^(b)^(a)These sequences were aligned to derive the corresponding primer.^(b)The nucleotide positions refer to the L. tropica tuf (EF-1) sequence fragment(SEQ ID NO. 526).^(c)These sequences are from the complementary DNA strand of the sequence of theoriginating fragment given in the Sequence Listing.^(d)The nucleotide positions refer to the E. coli tuf sequence fragment(SEQ ID NO. 78).^(e)The nucleotide positions refer to the B. cereus tuf sequence fragment(SEQ ID NO. 7). Originating DNA fragment SEQ ID Nucleotide SEQ ID NO.Nucleotide sequence NO. positionUniversal primers (bacteria) (continued) 6435′-ACT GGI GTI GAR ATG TTC CGY AA 1, 3, 4, 7, 12, 470-492^(b)13, 16, 49, 54, 72, 78, 85, 88, 91, 94, 98, 103, 108, 112, 115,116, 120, 121, 126, 128, 134, 136, 146, 154, 159, 179, 186,205, 209, 212, 224, 238^(a) 644^(c) 5′-ACG TCI GTI GTI CKG AAR TAG AAsame as SEQ 692-714^(b) ID NO. 643 643 5′-ACT GGI GTI GAR ATG TTC CGY AA1, 3, 4, 7, 12, 470-492^(b) 13, 16, 49, 54, 72, 78, 85, 88,91, 94, 98, 103, 108, 112, 115, 116, 120, 121, 126, 128, 134,136, 146, 154, 159, 179, 186, 205, 209, 212, 224, 238^(a) 645^(c)5′-ACG TCI GTI GTI CKG AAR TAR AA same as SEQ 692-714^(b) ID NO. 643 6465′-ATC GAC AAG CCI TTC YTI ATG SC 2, 13, 82 317-339^(d) 122, 145^(a)647^(c) 5′-ACG TCC GTS GTR CGG AAG TAG AAC TG 2, 13, 82 686-711^(d)122, 145^(a) 646 5′-ATC GAC AAG CCI TTC YTI ATG SC 2, 13, 82 317-339^(d)122, 145^(a) 648^(c) 5′-ACG TCS GTS GTR CGG AAG TAG AAC TG 2, 13, 82686-711^(d) 122, 145^(a)^(a)These sequences were aligned to derive the corresponding primer.^(b)The nucleotide positions refer to the E. coli tuf sequence fragmentSEQ ID NO. 78).^(c)These sequences are from the complementary DNA strand of the sequence of theoriginating fragment given in the Sequence Listing.^(d)The nucleotide positions refer to the A. meyeri tuf sequence fragment(SEQ ID NO. 2) Originating DNA fragment SEQ ID Nucleotide SEQ ID NO.Nucleotide sequence NO. positionUniversal primers (bacteria) (continued)  6495′-GTC CTA TGC CTC ARA CWC GIG AGC AC 8, 86, 141, 143^(a)  33-58^(b) 650^(c) 5′-TTA CGG AAC ATY TCA ACA CCI GT 8, 86, 141, 143^(a)473-495^(b)  636 5′-ACT GGY GTT GAI ATG TTC CGY AA 8, 86, 141, 143^(a)473-495^(b)  651^(c) 5′-TGA CGA CCA CCI TCY TCY TTY TTC A8, 86, 141, 143^(a) 639-663^(b) Universal primers (fungi) 19745′-ACA AGG GIT GGR MSA AGG AGA C 404, 405, 433, 443-464^(d)445, 898, 1268, 1276, 1986^(a) 1975^(c) 5′-TGR CCR GGG TGG TTR AGG ACG404, 405, 433, 846-866^(d) 445, 898, 1268, 1276, 1986^(a) 19765′-GAT GGA YTC YGT YAA ITG GGA 407-412, 286-306^(e) 414-426, 428-431,439, 443, 447, 448, 622, 624, 665, 1685, 1987-1990^(a) 1978^(c)5′-CAT CIT GYA ATG GYA ATC TYA AT same as SEQ 553-575^(e) ID NO. 19761977 5′-GAT GGA YTC YGT YAA RTG GGA same as SEQ 286-306^(e) ID NO. 19761979^(c) 5′-CAT CYT GYA ATG GYA ASC TYA AT same as SEQ 553-575^(e)ID NO. 1976 1981 5′-TGG ACA CCI SCA AGI GGK CYG 401-405, 281-301^(d)433, 435, 436, 438, 444, 445, 449, 453, 455, 457, 779,781-783, 785, 786, 788-790, 897-903, 1267-1272, 1274-1280,1282-1287, 1991-1998^(a) 1980^(c) 5′-TCR ATG GCI TCI AIR AGR GTY Tsame as SEQ 488-509^(d) ID NO. 1981^(a)These sequences were aligned to derive the corresponding primer.^(b)The nucleotide positions refer to the B. distasonis tuf sequence fragment(SEQ ID NO. 8).^(c)These sequences are from the complementary DNA strand of the sequence of theoriginating fragment given in the Sequence Listing.^(d)The nucleotide positions refer to the A. fumigatus tuf (EF-1) sequencefragment (SEQ ID NO. 404).^(e)The nucleotide positions refer to the C. albicans tuf (EF-1) sequence fragment(SEQ ID NO. 407). Originating DNA fragment SEQ ID Nucleotide SEQ ID NO.Nucleotide sequence NO. position Universal primers (fungi) (continued)1982 5′-TGG ACA CYI SCA AGI GGK CYG same as SEQ  281-301^(a) ID NO. 19811980^(b) 5′-TCR ATG GCI TCI AIR AGR GTY T same as SEQ  488-509^(a)ID NO. 1981 1983 5′-CYG AYT GCG CYA TIC TCA TCA same as SEQ  143-163^(a)ID NO. 1981 1980^(b) 5′-TCR ATG GCI TCI AIR AGR GTY T same as SEQ 488-509^(a) ID NO. 1981 1984 5′-CYG AYT GYG CYA TYC TSA TCA same as SEQ 143-163^(a) ID NO. 1981 1980^(b) 5′-TCR ATG GCI TCI AIR AGR GTY Tsame as SEQ  488-509^(a) ID NO. 1981 Sequencing primers  5565′-CGG CGC NAT CYT SGT TGT TGC  668^(c)  306-326  557^(b)5′-CCM AGG CAT RAC CAT CTC GGT G  668^(c) 1047-1068  6945′-CGG CGC IAT CYT SGT TGT TGC  668^(c)  306-326  557^(b)5′-CCM AGG CAT RAC CAT CTC GGT G  668^(c) 1047-1068  6645′-AAY ATG ATI ACI GGI GCI GCI CAR ATG GA  619^(c)  604-632  652^(b)5′-CCW AYA GTI YKI CCI CCY TCY CTI ATA  619^(c) 1482-1508  6645′-AAY ATG ATI ACI GGI GCI GCI CAR ATG GA  619^(c)  604-632  561^(b)5′-ACI GTI CGG CCR CCC TCA CGG AT  619^(c) 1483-1505  5435′-ATC TTA GTA GTT TCT GCT GCT GA 607    8-30  660^(b)5′-GTA GAA TTG AGG ACG GTA GTT AG 607  678-700  6585′-GAT YTA GTC GAT GAT GAA GAA TT 621  116-138  659^(b)5′-GCT TTT TGI GTT TCW GGT TTR AT 621  443-465  6585′-GAT YTA GTC GAT GAT GAA GAA TT 621  116-138  661^(b)5′-GTA GAA YTG TGG WCG ATA RTT RT 621  678-700  5585′-TCI TTY AAR TAY GCI TGG GT  665^(c)  157-176  559^(b)5′-CCG ACR GCR AYI GTY TGI CKC AT  665^(c) 1279-1301  8135′-AAT CYG TYG AAA TGC AYC ACG A  665^(c)  687-708  559^(b)5′-CCG ACR GCR AYI GTY TGI CKC AT  665^(c) 1279-1301^(a)The nucleotide positions refer to the A. fumigatus tuf (EF-1) sequencefragment (SEQ ID NO. 404).^(b)These sequences are from the complementary DNA strand of the sequence of theoriginating fragment given in the Sequence Listing.^(c)Sequences from databases. Originating DNA fragment SEQ ID NucleotideSEQ ID NO. Nucleotide sequence NO. positionSequencing primers (continued) 558 5′-TCI TTY AAR TAY GCI TGG GT 665^(a)  157-176 815^(b) 5′-TGG TGC ATY TCK ACR GAC TT  665^(a) 686-705 560 5′-GAY TTC ATY AAR AAY ATG ATY AC  665^(a)  289-311 559^(b)5′-CCG ACR GCR AYI GTY TGI CKC AT  665^(a) 1279-1301 6535′-GAY TTC ATI AAR AAY ATG AT  665^(a)  289-308 559^(b)5′-CCG ACR GCR AYI GTY TGI CKC AT  665^(a) 1279-1301 5585′-TCI TTY AAR TAY GCI TGG GT  665^(a)  157-176 655^(b)5′-CCR ATA CCI CMR ATY TTG TA  665^(a)  754-773 6545′-TAC AAR ATY KGI GGT ATY GG  665^(a)  754-773 559^(b)5′-CCG ACR GCR AYI GTY TGI CKC AT  665^(a) 1279-1301 6965′-ATI GGI CAY RTI GAY CAY GGI AAR AC  698^(a)   52-77 697^(b)5′-CCI ACI GTI CKI CCR CCY TCR CG  698^(a) 1132-1154 9115′-GAC GGM KKC ATG CCG CAR AC 853   22-41 914^(b)5′-GAA RAG CTG CGG RCG RTA GTG 853  700-720 9125′-GAC GGC GKC ATG CCG CAR AC 846   20-39 914^(b)5′-GAA RAG CTG CGG RCG RTA GTG 846  692-712 9135′-GAC GGY SYC ATG CCK CAG AC 843  251-270 915^(b)5′-AAA CGC CTG AGG RCG GTA GTT 843  905-925 9165′-GCC GAG CTG GCC GGC TTC AG 846  422-441 561^(b)5′-ACI GTI CGG CCR CCC TCA CGG AT  619^(a) 1483-1505 6645′-AAY ATG ATI ACI GGI GCI GCI CAR ATG GA  619^(a)  604-632 917^(b)5′-TCG TGC TAC CCG TYG CCG CCA T 846  593-614^(a)Sequences from databases.^(b)These sequences are from the complementary DNA strand of the sequence of theoriginating fragment given in the Sequence Listing.Originating DNA fragment SEQ ID Nucleotide SEQ ID NO.Nucleotide sequence NO. position Sequencing primers (continued) 12215′-GAY ACI CCI GGI CAY GTI GAY TT 1230^(a)  292-314 1226^(b)5′-GTI RMR TAI CCR AAC ATY TC 1230^(a) 2014-2033 12225′-ATY GAY ACI CCI GGI CAY GTI GAY TT 1230^(a)  289-314 1223^(b)5′-AYI TCI ARR TGI ARY TCR CCC ATI CC 1230^(a) 1408-1433 12245′-CCI GYI HTI YTI GAR CCI ATI ATG 1230^(a) 1858-1881 1225^(b)5′-TAI CCR AAC ATY TCI SMI ARI GGI AC 1230^(a) 2002-2027 12275′-GTI CCI YTI KCI GAR ATG TTY GGI TA 1230^(a) 2002-2027 1229^(b)5′-TCC ATY TGI GCI GCI CCI GTI ATC AT  698^(a)    4-29 12285′-GTI CCI YTI KCI GAR ATG TTY GGI TAY GC 1230^(a) 2002-2030 1229^(b)5′-TCC ATY TGI GCI GCI CCI GTI ATC AT  698^(a)    4-29 19995′-CAT GTC AAY ATT GGT ACT ATT GGT CAT GT 498-500, 502,   25-53^(d) 505, 506, 508,  619, 2004, 2005^(c) 2000^(b)5′-CCA CCY TCI CTC AMG TTG AAR CGT T same as SEQ  1133-1157^(d)ID NO. 1999 2001 5′-ACY ACI TTR ACI GCY GCY ATY AC same as SEQ  67-89^(d) ID NO. 1999 2003^(b) 5′-CAT YTC RAI RTT GTC ACC TGGsame as SEQ 1072-1092^(d) ID NO. 1999 20025′-CCI GAR GAR AGA GCI MGW GGT same as SEQ  151-171^(d) ID NO. 19992003^(b) 5′-CAT YTC RAI RTT GTC ACC TGG same as SEQ 1072-1092^(d)ID NO. 1999 ^(a)Sequences from databases.^(b)These sequences are from the complementary DNA strand of the sequence of theoriginating fragment given in the Sequence Listing.^(c)These sequences were aligned to derive the corresponding primer.^(d)The nucleotide positions refer to the C. albicans tuf sequence fragment(SEQ ID NO. 2004).

TABLE 40Specific and ubiquitous primers for nucleic acid amplification (atpD sequences).Originating DNA fragment SEQ ID Nucleotide SEQ ID NO.Nucleotide sequence NO. positionBacterial species: Acinetobacter baumannii 16905′-CAG GTC CTG TTG CGA CTG AAG AA 243 186-208 1691^(b)5′-CAC AGA TAA ACC TGA GTG TGC TTT C 243 394-418Bacterial species: Bacteroides fragilis 2134 5′-CGC GTG AAG CTT CTG TG929 184-200 2135^(b) 5′-TCT CGC CGT TAT TCA GTT TC 929 395-414Bacterial species: Bordetella pertussis 2180 5′-TTC GCC GGC GTG GGC1672^(c) 544-558 2181^(b) 5′-AGC GCC ACG CGC AGG 1672^(c) 666-680Bacterial species: Enterococcus faecium 16985′-GGA ATC AAC AGA TGG TTT ACA AA 292 131-153 1699^(b)5′-GCA TCT TCT GGG AAA GGT GT 292 258-277 17005′-AAG ATG CGG AAA GAA GCG AA 292 271-290 1701^(b)5′-ATT ATG GAT CAG TTC TTG GAT CA 292 439-461Bacterial species: Klebsiella pneumoniae 13315′-GCC CTT GAG GTA CAG AAT GGT AAT GAA GTT  317  88-118 1332^(b)5′-GAC CGC GGC GCA GAC CAT CA 317 183-203^(a)These sequences were aligned to derive the corresponding primer.^(b)These sequences are from the complementary DNA strand of the sequence of theoriginating fragment given in the Sequence Listing.^(c)Sequence from databases. Originating DNA fragment SEQ ID NucleotideSEQ ID NO. Nucleotide sequence NO. positionBacterial species: Streptococcus agalactiae  6275′-ATT GTC TAT AAA AAT GGC GAT AAG TC 379-383^(a)   42-67^(b)  625^(c)5′-CGT TGA AGA CAC GAC CCA AAG TAT CC 379-383^(a)  206-231^(b)  6285′-AAA ATG GCG ATA AGT CAC AAA AAG TA 379-383^(a)   52-77^(b)  625^(c)5′-CGT TGA AGA CAC GAC CCA AAG TAT CC 379-383^(a)  206-231^(b)  6275′-ATT GTC TAT AAA AAT GGC GAT AAG TC 379-383^(a)   42-67^(b)  626^(c)5′-TAC CAC CTT TTA AGT AAG GTG CTA AT 379-383^(a)  371-396^(b)  6285′-AAA ATG GCG ATA AGT CAC AAA AAG TA 379-383^(a)   52-77^(b)  626^(c)5′-TAC CAC CTT TTA AGT AAG GTG CTA AT 379-383^(a)  371-396^(b)Bacterial group: Campylobacter jejuni and C. cold. 21315′-AAG CMA TTG TTG TAA ATT TTG AAA G 1576, 1600,    7-31^(e)1849, 1863, 2139^(d,a) 2132^(c) 5′-TCA TAT CCA TAG CAA TAG TTC TA1576, 1600,   92-114^(e) 1849, 1863, 2139^(d,a)Bacterial genus: Bordetella sp.  8255′-ATG AGC ARC GSA ACC ATC GTT CAG TG 1672^(d)    1-26  826^(c)5′-TCG ATC GTG CCG ACC ATG TAG AAC GC 1672^(d) 1342-1367Fungal genus: Candida sp.  634 5′-AAC ACY GTC AGR RCI ATT GCY ATG GA460-472,  101-126^(f) 474-478^(a)  635^(c)5′-AAA CCR GTI ARR GCR ACT CTI GCT CT 460-472,  617-642^(f) 474-478^(a)^(a)These sequences were aligned to derive the corresponding primer.^(b)The nucleotide positions refer to the S. agalactiae atpD sequence fragment(SEQ ID NO. 380).^(c)These sequences are from the complementary DNA strand of the sequence of theoriginating fragment given in the Sequence Listing.^(d)Sequence from databases.^(e)The nucleotide positions refer to the C. jejuni atpD sequence fragment(SEQ ID NO. 1576).^(f)The nucleotide positions refer to the C. albicans atpD sequence fragment(SEQ ID NO. 460). Originating DNA fragment SEQ ID Nucleotide SEQ ID NO.Nucleotide sequence NO. position Universal primers 5625′-CAR ATG RAY GAR CCI CCI GGI GYI MGI ATG 243, 244, 262, 528-557^(b)264, 280, 284, 291, 297, 309, 311, 315, 317, 324, 329, 332,334-336, 339, 342, 343, 351, 356, 357, 364-366, 370, 375, 379, 393^(a)563^(c) 5′-GGY TGR TAI CCI ACI GCI GAI GGC AT 243, 244, 262, 687-712^(b)264, 280, 284, 291, 297, 309, 311, 315, 317, 324, 329, 332,334-336, 339, 342, 343, 351, 356, 357, 364-366, 370, 375, 379, 393^(a)564 5′-TAY GGI CAR ATG AAY GAR CCI CCI GGI AA 243, 244, 262, 522-550^(b)264, 280, 284, 291, 297, 309, 311, 315, 317, 324, 329, 332,334-336, 339, 342, 343, 351, 356, 357, 364-366, 370, 375, 379, 393^(a)565^(c) 5′-GGY TGR TAI CCI ACI GCI GAI GGD AT 243, 244, 262, 687-712^(b)264, 280, 284, 291, 297, 309, 311, 315, 317, 324, 329, 332,334-336, 339, 342, 343, 351, 356, 357, 364-366, 370, 375, 379, 393^(a)^(a)These sequences were aligned to derive the corresponding primer.^(b)The nucleotide positions refer to the K. pneumoniae atpD sequence fragment(SEQ ID NO. 317).^(c)These sequences are from the complementary DNA strand of the sequence of theoriginating fragment given in the Sequence Listing.Originating DNA fragment SEQ ID Nucleotide SEQ ID NO.Nucleotide sequence NO. position Universal primers (continued) 6405′-TCC ATG GTI TWY GGI CAR ATG AA 248, 284, 315, 513-535^(b)317, 343, 357, 366, 370, 379, 393^(a) 641^(c)5′-TGA TAA CCW ACI GCI GAI GGC ATA CG   248, 284, 315, 684-709^(b)317, 343, 357, 366, 370, 379, 393^(a) 6425′-GGC GTI GGI GAR CGI ACI CGT GA 248, 284, 315, 438-460^(b)317, 343, 357, 366, 370, 379, 393^(a) 641^(c)5′-TGA TAA CCW ACI GCI GAI GGC ATA CG   248, 284, 315, 684-709^(b)317, 343, 357, 366, 370, 379, 393^(a) Sequencing primers 5665′-TTY GGI GGI GCI GGI GTI GGI AAR AC 669^(d) 445-470 567^(c)5′-TCR TCI GCI GGI ACR TAI AYI GCY TG 669^(d) 883-908 5665′-TTY GGI GGI GCI GGI GTI GGI AAR AC 669^(d) 445-470 8145′-GCI GGC ACG TAC ACI GCC TG 666^(d) 901-920 5685′-RTI ATI GGI GCI GTI RTI GAY GT 669^(d)  25-47 567^(c)5′-TCR TCI GCI GGI ACR TAI AYI GCY TG 669^(d) 883-908 5705′-RTI RYI GGI CCI GTI RTI GAY GT 672^(d)  31-53 567^(c)5′-TCR TCI GCI GGI ACR TAI AYI GCY TG 669^(d) 883-908 5725′-RTI RTI GGI SCI GTI RTI GA 669^(d)  25-44 567^(c)5′-TCR TCI GCI GGI ACR TAI AYI GCY TG 669^(d) 883-908 5695′-RTI RTI GGI SCI GTI RTI GAT AT 671^(d)  31-53 567^(c)5′-TCR TCI GCI GGI ACR TAI AYI GCY TG 669^(d) 883-908 5715′-RTI RTI GGI CCI GTI RTI GAT GT 670^(d)  31-53 567^(c)5′-TCR TCI GCI GGI ACR TAI AYI GCY TG 669^(d) 883-908^(a)These sequences were aligned to derive the corresponding primer.^(b)The nucleotide positions refer to the K. pneumoniae atpD sequence fragment(SEQ ID NO. 317).^(c)These sequences are from the complementary DNA strand of the sequence of theoriginating fragment given in the Sequence Listing.^(d)Sequences from databases. Originating DNA fragment SEQ ID NucleotideSEQ ID NO. Nucleotide sequence NO. positionSequencing primers (continued)  700 5′-TIR TIG AYG TCG ART TCC CTC ARG 669^(a)   38-61  567^(b) 5′-TCR TCI GCI GGI ACR TAI AYI GCY TG  669^(a) 883-908  568 5′-RTI ATI GGI GCI GTI RTI GAY GT  669^(a)   25-47 573^(b) 5′-CCI CCI ACC ATR TAR AAI GC  666^(a) 1465-1484  5745′-ATI GCI ATG GAY GGI ACI GAR GG  666^(a)  283-305  573^(b)5′-CCI CCI ACC ATR TAR AAI GC  666^(a) 1465-1484  5745′-ATI GCI ATG GAY GGI ACI GAR GG  666^(a)  283-305  708^(b)5′-TCR TCC ATI CCI ARI ATI GCI ATI AT  666^(a) 1258-1283  6815′-GGI SSI TTY GGI ISI GGI AAR AC 685  694-716  682^(b)5′-GTI ACI GGY TCY TCR AAR TTI CCI CC 686 1177-1202  6815′-GGI SSI TTY GGI ISI GGI AAR AC 685  694-716  683^(b)5′-GTI ACI GGI TCI SWI AWR TCI CCI CC 685 1180-1205  6815′-GGI SSI TTY GGI ISI GGI AAR AC 685  694-716  6995′-GTI ACI GGY TCY TYR ARR TTI CCI CC 686 1177-1202  6815′-GGI SSI TTY GGI ISI GGI AAR AC 685  694-716  812^(b)5′-GTI ACI GGI TCY TYR ARR TTI CCI CC 685 1180-1205 12135′-AAR GGI GGI ACI GCI GCI ATH CCI GG  714^(a)  697-722 1212^(b)5′-CCI CCI RGI GGI GAI ACI GCW CC  714^(a) 1189-1211 12035′-GGI GAR MGI GGI AAY GAR ATG  709^(a)  724-744 1207^(b)5′-CCI TCI TCW CCI GGC ATY TC  709^(a)  985-1004 12045′-GCI AAY AAC ITC IWM YAT GCC  709^(a)  822-842 1206^(b)5′-CKI SRI GTI GAR TCI GCC A  709^(a)  926-944 12055′-AAY ACI TCI AWY ATG CCI GT  709^(a)  826-845 1207^(b)5′-CCI TCI TCW CCI GGC ATY TC  709^(a)  985-1004 22825′-AGR RGC IMA RAT GTA TGA  714^(a)   84-101 2284^(b)5′-TCT GWG TRA CIG GYT CKG AGA  714^(a) 1217-1237 22835′-ATI TAT GAY GGK ITT CAG AGG C  714^(a)  271-292 2285^(b)5′-CMC CIC CWG GTG GWG AWA C  714^(a) 1195-1213^(a)Sequences from databases.^(b)These sequences are from the complementary DNA strand of the sequence of theoriginating fragment given in the Sequence Listing.

TABLE 41Internal hybridization probes for specific detection of tuf sequences.Originating DNA fragment SEQ ID SEQ ID Nucleotide NO.Nucleotide sequence NO. position Bacterial species: Abiotrophia adiacens2170 5′-ACG TGA CGT TGA CAA ACC A 1715 313-331Bacterial species: Chlamydia pneumoniae 20895′-ATG CTG AAC TTA TTG ACC TT   20 136-155 20905′-CGT TAC TGG AGT CGA AAT G   20 467-485Bacterial species: Enterococcus faecalis  5805′-GCT AAA CCA GCT ACA ATC ACT CCA C 62-63, 607^(a) 584-608^(b)  6035′-GGT ATT AAA GAC GAA ACA TC 62-63, 607^(a) 440-459^(b) 11745′-GAA CGT GGT GAA GTT CGC 62-63, 607^(a) 398-415^(b)Bacterial species: Enterococcus faecium  6025′-AAG TTG AAG TTG TTG GTA TT 64, 608^(a) 426-445^(c)Bacterial species: Enterococcus gallinarum  6045′-GGT GAT GAA GTA GAA ATC GT 66, 609^(a) 419-438^(d)Bacterial species: Escherichia coli  579 5′-GAA GGC CGT GCT GGT GAG AA  78 503-522 2168 5′-CAT CAA AGT TGG TGA AGA AGT TG   78 409-431Bacterial species: Neisseria gonorrhoeae 2166 5′-GAC AAA CCA TTC CTG CTG 126 322-339^(e) Fungal species: Candida albicans  5775′-CAT GAT TGA ACC ATC CAC CA 407-411^(a) 406-425^(f)Fungal species: Candida dubliniensis  578 5′-CAT GAT TGA AGC TTC CAC CA412, 414-415^(a) 418-437^(g)^(a)These sequences were aligned to derive the corresponding primer.^(b)The nucleotide positions refer to the E. faecalis tuf sequence fragment(SEQ ID NO. 607).^(c)The nucleotide positions refer to the E. faecium tuf sequence fragment(SEQ ID NO. 608).^(d)The nucleotide positions refer to the E. gallinarum tuf sequence fragment(SEQ ID NO. 609).^(e)The nucleotide positions refer to the N. gonorrhoeae tuf sequence fragment(SEQ ID NO. 126).^(f)The nucleotide positions refer to the C. albicans tuf(EF-1) sequence fragment(SEQ ID NO. 408).^(g)The nucleotide positions refer to the C. dubliniensis tuf(EF-1) sequence fragment(SEQ ID NO. 414). Originating DNA fragment SEQ ID SEQ ID Nucleotide NO.Nucleotide sequence NO. positionBacterial species: Haemophilus influenzae  5815′-ACA TCG GTG CAT TAT TAC GTG G  610^(a) 551-572Bacterial species: Mycoplasma pneumoniae 2095 5′-CGG TCG GGT TGA ACG TGG2097^(a) 687-704 Bacterial species: Staphylococcus aureus  5845′-ACA TGA CAC ATC TAA AAC AA 176-180^(b) 369-388^(c)  5855′-ACC ACA TAC TGA ATT CAA AG 176-180^(b) 525-544^(c)  5865′-CAG AAG TAT ACG TAT TAT CA 176-180^(b) 545-564^(c)  5875′-CGT ATT ATC AAA AGA CGA AG 176-180^(b) 555-574^(c)  5885′-TCT TCT CAA ACT ATC GTC CA 176-180^(b) 593-612^(c)Bacterial species: Staphylococcus epidermidis  5895′-GCA CGA AAC TTC TAA AAC AA 185, 611^(b) 445-464^(d)  5905′-TAT ACG TAT TAT CTA AAG AT 185, 611^(b) 627-646^(d)  5915′-TCC TGG TTC TAT TAC ACC AC 185, 611^(b) 586-605^(d)  5925′-CAA AGC TGA AGT ATA CGT AT 185, 611^(b) 616-635^(d)  5935′-TTC ACT AAC TAT CGC CCA CA 185, 611^(b) 671-690^(d)Bacterial species: Staphylococcus haemolyticus  5945′-ATT GGT ATC CAT GAC ACT TC 186, 188-190^(b) 437-456^(e)  5955′-TTA AAG CAG ACG TAT ACG TT 186, 188-190^(b) 615-634^(e)Bacterial species: Staphylococcus hominis  5965′-GAA ATT ATT GGT ATC AAA GA 191, 193-196^(b) 431-450^(f)  5975′-ATT GGT ATC AAA GAA ACT TC 191, 193-196^(b) 437-456^(f)  5985′-AAT TAC ACC TCA CAC AAA AT 191, 193-196^(b) 595-614^(f)^(a)Sequences from databases.^(b)These sequences were aligned to derive the corresponding probe.^(c)The nucleotide positions refer to the S. aureus tuf sequence fragment(SEQ ID NO. 179).^(d)The nucleotide positions refer to the S. epidermidis tuf sequence fragment(SEQ ID NO. 611).^(e)The nucleotide positions refer to the S. haemolyticus tuf sequence fragment(SEQ ID NO. 186).^(f)The nucleotide positions refer to the S. hominis tuf sequence fragment(SEQ ID NO. 191). Originating DNA fragment SEQ ID SEQ ID Nucleotide NO.Nucleotide sequence NO. positionBacterial species: Staphylococcus saprophyticus  5995′-CGG TGA AGA AAT CGA AAT CA 198-200^(a) 406-425^(b)  6005′-ATG CAA GAA GAA TCA AGC AA 198-200^(a) 431-450^(b)  6015′-GTT TCA CGT GAT GAT GTA CA 198-200^(a) 536-555^(b)  6955′-GTT TCA CGT GAT GAC GTA CA 198-200^(a) 563-582^(b)Bacterial species: Streptococcus agalactiae   582^(c)5′-TTT CAA CTT CGT CGT TGA CAC GAA CAG T 207-210^(a) 404-431^(d)  583^(c) 5′-CAA CTG CTT TTT GGA TAT CTT CTT TAA TAC CAA CG  207-210^(a)433-467^(d) 1199 5′-GTA TTA AAG AAG ATA TCC AAA AAG C 207-210^(a)438-462^(d) Bacterial species: Streptococcus pneumoniae 12015′-TCA AAG AAG AAA CTA AAA AAG CTG T 971, 977, 513-537^(e) 979, 986^(a)Bacterial species: Streptococcus pyogenes 12005′-TCA AAG AAG AAA CTA AAA AAG CTG T 1002 473-497Bacterial group: Enterococcus casseliflavus-flavescens-gallinarum group 620 5′-ATT GGT GCA TTG CTA CGT 58, 65, 66^(a) 527-544^(f) 11225′-TGG TGC ATT GCT ACG TGG 58, 65, 66^(a) 529-546^(f)Bacterial group: Enterococcus sp., Gemella sp., A. adiacens 21725′-GTG TTG AAA TGT TCC GTA AA 58-62, 67-71, 477-496^(g) 87-88, 607-609,727, 871 1715, 1722^(a)^(a)These sequences were aligned to derive the corresponding primer.^(b)The nucleotide positions refer to the S. saprophyticus tuf sequence fragment(SEQ ID NO. 198).^(c)These sequences are from the complementary DNA strand of the sequence of the originating fragment given in the Sequence Listing.^(d)The nucleotide positions refer to the S. agalactiae tuf sequence fragment(SEQ ID NO. 209).^(e)The nucleotide positions refer to the S. pneumoniae tuf sequence fragment(SEQ ID NO. 986).^(f)The nucleotide positions refer to the E. flavescens tuf sequence fragment(SEQ ID NO. 65).^(g)The nucleotide positions refer to the E. faecium tuf sequence fragment(SEQ ID NO. 608). Originating DNA fragment SEQ ID SEQ ID Nucleotide NO.Nucleotide sequence NO. position Bacterial genus: Gemella 21715′-TCG TTG GAT TAA CTG AAG AA 87, 88^(a) 430-449^(b)Bacterial genus: Staphylococcus sp.  605 5′-GAA ATG TTC CGT AAA TTA TT176-203^(a) 403-422^(c)  606 5′-ATT AGA CTA CGC TGA AGC TG 176-203^(a)420-439^(c) 1175 5′-GTT ACT GGT GTA GAA ATG TTC 176-203^(a) 391-411^(c)1176 5′-TAC TGG TGT AGA AAT GTT C 176-203^(a) 393-411^(c)Bacterial genus: Streptococcus sp. 1202 5′-GTG TTG AAA TGT TCC GTA AAC A206-231, 971, 466-487^(d) 977, 979,  982-986^(a)Fungal species: Candida albicans 1156 5′-GTT GAA ATG CAT CAC GAA CAA TT407-412, 624^(a) 680-702^(e)Fungal group: Candida albicans and C. tropicalis 11605′-CGT TTC TGT TAA AGA AAT TAG AAG 407-412, 748-771^(e) 429, 624^(a)Fungal species: Candida dubliniensis 11665′-ACG TTA AGA ATG TTT CTG TCA A 414-415^(a) 750-771^(f) 11685′-GAA CAA TTG GTT GAA GGT GT 414-415^(a) 707-726^(f)Fungal species: Candida glabrata 1158 5′-AAG AGG TAA TGT CTG TGG T 417781-799 1159 5′-TGA AGG TTT GCC AGG TGA 417 718-735Fungal species: Candida krusei 1161 5′-TCC AGG TGA TAA CGT TGG 422720-737^(a)These sequences were aligned to derive the corresponding primer.^(b)The nucleotide positions refer to the G. haemolysans tuf sequence fragment(SEQ ID NO. 87).^(c)The nucleotide positions refer to the S. aureus tuf sequence fragment(SEQ ID NO. 179).^(d)The nucleotide positions refer to the S. pneumoniae tuf sequence fragment(SEQ ID NO. 986).^(e)The nucleotide positions refer to the C. albicans tuf(EF-1) sequence fragment(SEQ ID NO. 408).^(f)The nucleotide positions refer to the C. dubliniensis tuf(EF-1) sequence fragment(SEQ ID NO. 414). Originating DNA fragment SEQ ID SEQ ID Nucleotide NO.Nucleotide sequence NO. positionFungal group: Candida lusitaniae and C. guillermondii 11625′-CAA GTC CGT GGA AAT GCA 418, 424^(a) 682-699^(b)Fungal species: Candida parapsilosis 11575′-AAG AAC GTT TCA GTT AAG GAA AT 426 749-771Fungal species: Candida zeylanoides 1165 5′-GGT TTC AAC GTG AAG AAC 432713-730 Fungal genus: Candida sp. 1163 5′-GTT GGT TTC AAC GTT AAG AAC407-412, 414- 728-748^(c) 415, 417, 418, 422, 429^(a) 11645′-GGT TTC AAC GTC AAG AAC 413, 416, 420, 740-757^(b) 421, 424, 425,426, 428, 431^(a) 1167 5′-GTT GGT TTC AAC GT 406-426, 428- 728-741^(c)432, 624^(a)^(a)These sequences were aligned to derive the corresponding primer.^(b)The nucleotide positions refer to the C. lusitaniae tuf(EF-1) sequence fragment(SEQ ID NO. 424).^(c)The nucleotide positions refer to the C. albicans tuf(EF-1) sequence fragment(SEQ ID NO. 408).

TABLE 42Strategy for the selection of amplification/sequencing primers from atpD (F-type) sequences.SEQ ID Accession23                         49   443                           472   881                           910NO.: #: B. cepaciaAGTgCAT CGGCGCCGTT ATCGACGTGG...TGTTCG GCGGTGCTGG CGTGGGCAAG ACCG...TCCA GGCCGTGT ACGTCCCTGC GGACGACT2346 X76877 B. pertussisAGTgCAT CGGCGCCGTG GTGGATATTC...TGTTCG GCGGCGCCGG CGTGGGCAAG ACCG...TCCA GGCCGTGT ACGTGCCTGC CGACGACT2347 Genome project P. aeruginosaAAATTAT CGGCGCCGTG ATCGACGTGG...TGTTCG GCGGCGCCGG CGTGGGCAAG ACCG...TCCA GGCCGTAT ACGTTCCCGC GGACGACC2348 Genome project E. coliAGGTAAT CGGCGCCGTA GTTGACGTCG...TGTTCG GTGGTGCGGG TGTAGGTAAA ACCG...TACA GGCAGTAT ACGTACCTGC GGATGACT2349 J01594 N. gonorrhoeaeAAATTAT CGGTGCGGTT GTTGACGTGG...TGTTCG GCGGTGCCGG TGTGGGTAAA ACCG...TCCA AGCCGTAT ATGTACCTGC GGATGACT2350 Genome project M. thermoaceticaAGGTTAT TGGCCCGGTG GTTGACGTCG...TCTTCG GCGGCGCCGG GGTCGGCAAG ACGG...TGCA AGCTATCT ATGTGCCGGC CGACGACC2351 U64318 S. aurantiacaAGGTTcT CGGTCCCGTG ATTGACGTGG...TGTTCG GCGGCGCCGG CGTGGGCAAG ACGG...TGCA GGCCATCT ACGTGCCCGC CGACGACC2352 X76879 M. tuberculosisGGGTCAC TGGGCCCGTC GTCGACGTCG...TGTTCG GCGGTGCCGG GGTGGGCAAG ACGG...TGCA AGCCGTCT ACGTGCCCGC CGACGACT2353 Z73419 B. fragilisAGGTAAT TGGCCCTGTG GTCGATGTGT...TGTTTG GCGGGGCCGG AGTGGGTAAA ACTG...TGCA GGCTGTTT ACGTACCGGC TGATGACT2354 M22247 C. lyticaAAATTAT TGGCCCAGTT ATAGATGTGG...TATTTG GAGGTGCCGG AGTAGGTAAA ACAG...TACA GGCGGTTT ACGTACCTGC GGATGATT 672 M22535 A. woodiiAGGTTAT TGGACCAGTA GTCGATGTTA...TTTTCG GTGGTGCCGG AGTTGGTAAA ACCG...TTCA GGCCGTTT ACGaTCCAGC CGATGACT2356 U10505 C. acetobutylicumAGGTAAT AGGACCTGTT GTGGATATTA...TGTTCG GTGGTGCCGG TGTTGGTAAA ACAG...TTCA GGCTGTAT ATGTTCCTGC TGATGACC 671 AF101055 M. pneumoniaeAAGTGAT TGGCCCGGTA GTTGATGTCA...TATTTG GTGGTGCTGG TGTTGGTAAA ACGG...TGCA AGCGATCT ATGTGCCAGC TGATGACT2357 U43738 H. pyloriAGGTTtT AGGCCCGGTG GTAGATGTGG...TGTTTG GTGGGGCTGG CGTAGGCAAA ACGG...TTCA AGCGGTGT ATGTGCCAGC AGACGACT 670 AF004014 Selected sequences   RTIAT IGGIGCIGTI RTIGAYGT  568for universal primers  RTIRY IGGICCIGTI RTIGAYGT  570 RTIRT IGGISCIGTI RTIGA  572  RTIRT IGGISCIGTI RTIGATAT  569 RTIRT IGGICCIGTI RTIGATGT  571                                  TTYG GIGGIGCIGG IGTIGGIAAR AC  566Selected sequence                                                                      CA RGCIRTIT AYGTICCIGC IGAYGA 567 for universal primer^(a) The sequence numbering refers to theEscherichia coli atpD gene fragment (SEQ ID NO. 669). Nucleotides incapitals are identical to the selected sequences or match thosesequences. Mismatches are indicated by lower-case letters. Dots indicategaps in the sequences displayed. “R” “Y” “M” “K” “W” and “S” designatenucleotide positions which are degenerated. “R” stands for A or G; “Y”stands for C or T; “M” stands for A or C; “K” stands for G or T; “W”stands for A or T; “S” stands for C or G. “I” stands for inosine whichis a nucleotide analog that can bind to any of the four nucleotides A,C, G or T. ^(a)This sequence is the reverse-complement of the selectedprimer.

TABLE 43Strategy for the selection of universal amplification/sequencingprimers from atpD (V-type) sequences. SEQ ID691                          719   1177                           1208NO.: E. hiraeCC AGGTCCGTTT GGTGCAGGGA AGACAGT...TCTGGTGGAg ATaTCtctGA ACCAGTGACT CA685 H. salinarumCC GGGGCCGTTC GGGTCCGGGA AGACGGT...CCCGGCGGGg ACTTCtccGA GCCGGTCACC CA687 T. thermophilusCC TGGGCCCTTC GGCAGCGGCA AGACCGT...CCGGGCGGCg ACaTgtccGA GCCCGTGACC CA693 HumanCC TGGGGCCTTC GGATGTGGCA AGACTGT...CCCGGTGGAg ACTTCtcAGA tCCCGTGACG AC688 T. congolenseCC TGGCGCGTTT GGATGCGGAA AGACGGT...CCTGGAGGTg ACTTTtctGA cCCAGTGACG TC692 P. falciparumCC TGGTGCATTT GGTTGTGGAA AAACTTG...CCAGGTGGTg ATTTCtctGA cCCTGTAACT AC689 C. pneumoniaeCC AGGACCTTTT GGTGCAGGGA AAACAGT...GCAGGAGGAA ACTTTGAAGA ACCAGTCACT CA686 Selected sequences     GGISSITTY GGIISIGGIA ARACfor universal primers 681 Selected sequences                                      GGIGGIA AYTTYGARGA RCCIGTIAC 682for universal primers^(a)                                      GGIGGIG AYWTIWSIGA ICCIGTIAC 683The sequence numbering refers to the Enterococcus hirae atpD genefragment (SEQ ID NO. 685). Nucleotides in capitals are identical to theselected sequences or match those sequences. Mismatches for SEQ ID NOs.681 and 682 are indicated by lower-case letters. Mismatches for SEQ IDNO. 683 are indicated by underlined nucleotides. Dots indicate gaps inthe sequences displayed. “R” “Y” “M” “K” “W” and “S” designatenucleotide positions which are degenerated. “R” stands for A or G; “Y”stands for C or T; “M” stands for A or C; “K” stands for G or T; “W”stands for A or T; “S” stands for C or G. “I” stands for inosine whichis a nucleotide analog that can bind to any of the four nucleotides A,C, G or T. ^(a)These sequences are the reverse-complement of theselected primers.

TABLE 44Strategy for the selection of universal amplification/sequencingprimers from tuf (M) sequences (organelle origin). SEQ Acces- ID sion601                                635   1479                            1511NO.:  #: C.AAGAA CATGATCACC GGTaCCtCCC AGgctGACTG...CGCcgTCcGA GAcatGcGAC AGACcGTTGc CGT2358 U81803 neoformans ^(a) S.AAGAA CATGATTACT GGTaCTtCTC AAgctGACTG...CGCTgTCAGA GAcatGaGAC AAACTGTcGc TGT 665 X00779 cerevisiae ^(a) O. volvulus ^(a)AAGAA TATGATCACA GGTaCTtCTC AGgctGACTG...TGCTgTGcGt GAtatGaGAC AAACaGTTGc GGT2359 M64333 Human^(a)AAAAA CATGATTACA GGGaCAtCTC AGgctGACTG...TGCTgTTcGt GAtatGaGAC AGACaGTTGc TGT2360 X03558 G. max B1^(b)AAGAA CATGATCACC GGCGCTGCCC AGATGGACGG...TGCTATTAGA GAAGGAGGCA AAACTGTTGG AGC2361 Y15107 G. max B2^(b)AAAAA CATGATCACC GGCGCCGCCC AGATGGACGG...TGCTATTAGA GAAGGAGGCA AAACTGTTGG AGC2362 Y15108 E. coli ^(c)AAAAA CATGATCACC GGTGCTGCTC AGATGGACGG...CGCaATCcGt GAAGGCGGCC GTACcGTTGG CGC  78 — S.AAGAA CATGATCACC GGTGCCGCCC AGATGGACGG...CGCcATCcGt GAGGGTGGTC GTACcGTgGG CGC2363 AF007125 aureofaciens ^(c) E. tenella ^(b)AAAAA TATGATTACA GGAGCAGCAC AAATGGATGG...TGCTATAAGA GAAGGAGGAA AAACTATAGG AGC2364 AI755521 T. gondii ^(b)AAGAA TATGATTACT GGAGCCGCAC AAATGGATGG...TGCTATTAGA GAAGGAGGTC GTACTATAGG AGC2365 Y11431 S.AAGAA TATGATTACC GGTGCTGCTC AAATGGATGG...CAATATCAGA GAGGGTGGAA GAACTGTTGG TAC 619 K00428 cerevisiae ^(b) A. thaliana ^(b)AAAAA TATGATTACT GGAGCTGCGC AAATGGATGG...TGCctTAAGG GAAGGAGGTA GAACaGTTGG AGC2366 X89227 Selected    AA YATGATIACI GGIGCIGCIC ARATGGA  664sequence for universal  primer Selected                                              TATIAGR GARGGIGGIM RIACTRTWGG^(d) 652 sequences for                                             ATCCGT GAGGGYGGCC GITCIGT^(d) 561 universal  primers The sequence numbering refers to theSaccharomyces cerevisiae tuf (M) gene (SEQ ID NO. 619). Nucleotides incapitals are identical to the selected sequences or match thosesequences. Mismatches for SEQ ID NOs. 652 and 664 are indicated bylower-case letters. Mismatches for SEQ ID NO. 561 are indicated byunderlined nucleotides. Dots indicate gaps in the sequences displayed.“R” “Y” “M” “K” “W” and “S” designate nucleotide positions which aredegenerated. “R” stands for A or G; “Y” stands for C or T; “M” standsfor A or C; “K” stands for G or T; “W” stands for A or T; “S” stands forC or G. “I” stands for inosine which is a nucleotide analog that canbind to any of the four nucleotides A, C, G or T. ^(a)This sequencerefers to tuf(EF-1) gene. ^(b)This sequence refers to tuf (M) ororganelle gene. ^(c)This sequence refers to tuf gene from bacteria.^(d)These sequences are the reverse-complement of the selected primers.

TABLE 45Strategy for the selection of eukaryotic sequencing primers from tuf (EF-1) sequences.SEQ ID Accession154                       179   286                          314  NO.: #: S. cerevisiaeGG TTCTTTCAAG TACGCTTGGG TTTT...AGAGA TTTCATCAAG AACATGATTA CTGG...  665X00779 B. hominisGG CTCCTTCAAG TACGCGTGGG TGCT...CGTGA CTTCATCAAG AACATGATCA CGGG... 2367D64080 C. albicansGG TTCTTTCAAA TACGCTTGGG TCTT...AGAGA TTTCATCAAG AACATGATCA CTGG... 2368M29934 C. neoformansTC TTCTTTCAAG TACGCTTGGG TTCT...CGAGA CTTCATCAAG AACATGATCA CCGG... 2369U81803 E. histolyticaGG ATCATTCAAA TATGCTTGGG TCTT...AGAGA TTTCATTAAG AACATGATTA CTGG... 2370M92073 G. lambliaGG CTCCTTCAAG TACGCGTGGG TCCT...CGCGA CTTCATCAAG AACATGATCA CGGG... 2371D14342 H. capsulatumAA ATCCTTCAAA TATGCGTGGG TCCT...CGTGA CTTCATCAAG AACATGATCA CTGG... 2372U14100 HumanGG CTCCTTCAAG TATGCCTGGG TCTT...AGAGA CTTtATCAAA AACATGATTA CAGG... 2373X03558 L. braziliensisGC GTCCTTCAAG TACGCGTGGG TGCT...CGCGA CTTCATCAAG AACATGATCA CCGG... 2374U72244 O. volvulusGG CTCATTTAAA TATGCTTGGG TATT...CGTGA TTTCATTAAG AATATGATCA CAGG... 2375M64333 P. bergheiGG TagTTTCAAA TATGCATGGG TTTT...AAAcA TTTtATTAAA AATATGATTA CTGG... 2376AJ224150 P. knowlesiGG AagTTTTAAG TACGCATGGG TGTT...AAGGA TTTCATTAAA AATATGATTA CCGG... 2377AJ224153 S. pombeGG TTCCTTCAAG TACGCCTGGG TTTT...CGTGA TTTCATCAAG AACATGATTA CCGG... 2378U42189 T. cruziTC TTCTTTCAAG TACGCGTGGG TCTT...CGCGA CTTCATCAAG AACATGATCA CGGG... 2379L76077 Y. lipolyticaGG TTCTTTCAAG TACGCTTGGG TTCT...CGAGA TTTCATCAAG AACATGATCA CCGG... 2380AF054510 Selected     TCITTYAAR TAYGCITGGG T  558 sequences for                                   GA YTTCATYAAR AAYATGATYA C  560amplification                                    GA YTTCATIAAR AAYATGAT 653 primersThe sequence numbering refers to the Saccharomyces cerevisiae tuf (EF-1) gene fragment (SEQ ID NO. 665). Nucleotides in capitals are identical to the selected sequences SEQ ID NOs. 558, 560 or 653, ormatch those sequences. Mismatches for SEQ ID no. 558 and 560 are indicated by lower-case letters.Mismatches for SEQ ID NO. 653 are indicated by underlined nucleotides.Dots indicate gaps in the sequences displayed. “R” “Y” “M” “K” “W”and “S” designate nucleotide positions which are degenerated. “R”stands for A or G; “Y” stands for C or T; “M” stands for A or C; “K”stands for G or T; “W” stands for A or T; “S” stands for C or G. “I”stands forinosine which is a nucleotide analog that can bind to any of the four nucleotides A, C, G or T.SEQ ID Accession   751                      776   1276                        1304  NO.: #: S. cerevisiae...GTTTACAA GATCGGTGGT AgTGGTAC...GACATG AGACAAACTG TCGCTGTCGG TGT  665X00779 B. hominis...GTGTACAA GATTGGCGGT ATTGGTAC...GATATG AGACAGACTG TCGCTGTCGG TAT 2381D64080 C. albicans...GTTTACAA GATCGGTGGT ATTGGTAC...GATATG AGACAAACCG TTGCTGTtGG TGT 2382M29934 C. neoformans...GTCTACAA GATCGGTGGT ATCGGCAC...GACATG CGACAGACCG TTGCCGTtGG TGT 2383U81803 E. histolytica...GTTTACAA GATTTcAGGT ATTGGAAC...GATATG AaACAAACCG TTGCTGTtGG AGT 2384M92073 G. lamblia ...GTCTACAA GATCTcGGGc gTCGGGAC...~~~~~~ ~~~~~~~~~~~~~~~~~~~~ ~~~ 2385 D14342 H. capsulatum...GTGTACAA AATCTcTGGT ATTGGCAC...GACATG AGACAAACCG TCGCTGTCGG TGT 2386U14100 Human...GTCTACAA AATTGGTGGT ATTGGTAC...GATATG AGACAGACAG TTGCgGTgGG TGT 2387X03558 L. braziliensis...GTGTACAA GATCGGCGGT ATCGGCAC...GACATG CGCagAACGG TCGCCGTCGG CAT 2388U72244 O. volvulus...GTTMACAA AATTGGAGGT ATTGGAAC...GATATG AGACAAACAG TTGCTGTtGG CGT 2389M64333 P. berghei...GTATACAA AATTGGTGGT ATTGGTAC...GATATG AGACAAACAA TTGCTGTCGG TAT 2390AJ224150 P. knowlesi...GTATACAA AATCGGTGGT ATTGGTAC...GATATG AGACAAACCA TTGCTGTCGG TAT 2391AJ224153 S. pombe...GTTTACAA GATCGGTGGT ATTGGTAC...GACATG CGTCAAACCG TCGCTGTCGG TGT 2392U42189 T. cruzi...GTGTACAA GATCGGCGGT ATCGGCAC...GACATG CGCCAGACGG TCGCCGTCGG CAT 2393L76077 Y. lipolytica...GTCTACAA GATCGGTGGT ATCGGCAC...GACATG CGACAGACCG TTGCTGTCGG TGT 2394AF054510 Selected       TACAA RATYKGIGGT ATYGG  654 sequence foramplification primer Selected       TACAA RATYKGIGGT ATYGG  655sequences for                                     ATG MGICARACIR TYGCYGTCGG  559amplification primers^(a)The sequence numbering refers to the Saccharomyces cerevisiae tuf (EF-1) gene fragment (SEQ ID NO. 665). Nucleotides in capitals are identical to the selected sequences or match those sequences. Mismatchesare indicated by lower-case letters. “~”indicate incomplete sequence data.Dots indicate gaps in the sequences displayed. “R” “Y” “M” “K” “W”and “S” designate nucleotide positions which are degenerated. “R”stands for A or G; “Y” stands for C or T; “M” stands for A or C; “K”stands for G or T; “W” stands for A or T; “S” stands for C or G. “I”stands forinosine which is a nucleotide analog that can bind to any of the four nucleotides A, C, G or T.^(a)This sequences are the reverse-complement of the selected primers.

TABLE 46 Strategy for the selection of Streptococcus agalactiae-specificamplification primers from tuf sequences. SEQ ID Accession305                           334   517                       542 NO.: #: S. agalactiaeCCAGAA CGTGATACTG ACAAACCTTT ACTT...GGAC AACGTTGGTG TTCTTCTTCG TG  207 —S. agalactiaeCCAGAA CGTGATACTG ACAAACCTTT ACTT...GGAC AACGTTGGTG TTCTTCTTCG TG  208 —S. agalactiaeCCAGAA CGTGATACTG ACAAACCTTT ACTT...GGAC AACGTTGGTG TTCTTCTTCG TG  209 —S. agalactiaeCCAGAA CGTGATACTG ACAAACCTTT ACTT...GGAC AACGTTGGTG TTCTTCTTCG TG  210 —S. anginosusCCAGAA CGTGAcACTG ACAAACCaTT gCTT...AGAt AACGTaGGgG TTCTTCTTCG TG  211 —S. anginosusCCAGAA CGTGATACTG ACAAACCaTT gCTT...AGAt AACGTaGGgG TTCTTCTTCG TG  221 —S. bovisCCAaAA CGTGATACTG ACAAACCaTT gCTT...GGAt AACGTTGGTG TTCTTCTTCG TG  212 —S. gordoniiCCAGAA CGTGAcACTG ACAAACCaTT gCTT...AGAt AAtGTaGGTG TTCTTCTTCG TG  223 —S. mutansCCAGAA CGTGATACTG ACAAgCCgcT cCTT...GGAt AAtGTTGGTG TTCTcCTTCG TG  224 —S. pneumoniaeCCAGAA CGTGAcACTG ACAAACCaTT gCTT...AGAt AACGTaGGTG TTCTTCTTCG TG  145^(a) S. sanguinisCCAGAA CGcGATACTG ACAAgCCaTT gCTT...GGAC AACGTaGGTG TgCTTCTcCG TG  227 —S. sobrinusCCAaAA CGcGATACTG AtAAgCCaTT gCTT...AGAt AACGTTGGTG TgCTTCTTCG TG  228 —B. cepaciaCCGGAg CGTGcagtTG ACggcgCgTT cCTG...CGAC AACGTTGGTa TcCTgCTgCG cG   16 —B. fragilisCCTccg CGcGATgtTG AtAAACCTTT ctTG...TGAC AACGTaGGTc TgtTgCTTCG TG 2395P33165 B. subtilisCCAGAA CGcGAcACTG AaAAACCaTT caTG...TGAC AACaTTGGTG ccCTTCTTCG cG 2396Z99104 C. diphtheriaeCCAGAg CGTGAgACcG ACAAgCCaTT cCTC...CGAC AACtgTGGTc TgCTTCTcCG TG  662 —C. trachomatisCCAGAA aGaGAaAtTG ACAAgCCTTT cTTA...AGAg AAtGTTGGat TgCTcCTcaG aG   22 —E. coliCCAGAg CGTGcgAtTG ACAAgCCgTT cCTg...TGAg AACGTaGGTG TTCTgCTgCG TG   78 —G. vaginalisCCAact CacGATctTG ACAAgCCaTT cTTg...CGAt RACacTGGTc TTCTTCTcCG cG  135^(a) S. aureusCCAGAA CGTGATtCTG ACAAACCaTT cATg...TGAC AACaTTGGTG catTatTaCG TG  179 —Selected sequence    GAA CGTGATACTG ACAAACCTTT A  549 for species-specific primer Selected sequence                                       C AACGTTGGTG TTCTTCTTC  550for species- specific primer^(b) The sequence numbering refers to theStreptococcus agalactiae tuf gene fragment (SEQ ID NO. 209). Nucleotidesin capitals are identical to the selected sequences or match thosesequences. Mismatches are indicated by lower-case letters. Dots indicategaps in the sequences displayed. “R” “Y” “M” “K” “W” and “S” designatenucleotide positions which are degenerated. “R” stands for A or G; “Y”stands for C or T; “M” stands for A or C; “K” stands for G or T; “W”stands for A or T; “S” stands for C or G. “I” stands for inosine whichis a nucleotide analog that can bind to any of the four nucleotides A,C, G or T. ^(a)The SEQ ID NO. refers to previous patent publicationWO98/20157. ^(b)This sequence is the reverse-complement of the selectedprimer.

TABLE 47 Strategy for the selection of Streptococcus agalactiae-specifichybridization probes from tuf sequences.401                            431 433                                    470SEQ ID NO.: Accession #: S. acidominimusGGTACTGT TaaaGTtAAt GACGAAGTTG AAATCGTTGG TATcAAAGAc GAaATCtctA AAGCAGTTGT TA 206 S. agalactiaeGGTACTGT TCGTGTCAAC GACGAAGTTG AAATCGTTGG TATTAAAGAA GATATCCAAA AAGCAGTTGT TA 209 S. agalactiaeGGTACTGT TCGTGTCAAC GACGAAGTTG AAATCGTTGG TATTAAAGAA GATATCCAAA AAGCAGTTGT TA2397 S. agalactiaeGGTACTGT TCGTGTCAAC GACGAAGTTG AAATCGTTGG TATTAAAGAA GATATCCAAA AAGCAGTTGT TA 207 S. agalactiaeGGTACTGT TCGTGTCAAC GACGAAGTTG AAATCGTTGG TATTAAAGAA GATATCCAAA AAGCAGTTGT TA 210 S. agalactiaeGGTACTGT TCGTGTCAAC GACGAAGTTG AAATCGTTGG TATTAAAGAA GATATCCAAA AAGCAGTTGT TA 208 S. anginosusGGTACTGT TaaaGTCAAC GACGAAGTTG AAATCGTTGG TATccgtGAt GAaATCCAAA AAGCAGTTGT TA 211 S. anginosusGGTACTGT TaaaGTCAAC GAtGAAGTTG AAATCGTTGG TATccgcGAg GAaATCCAAA AAGCAGTTGT TA 221 S. bovisGGTACTGT TaaaGTCAAC GACGAAGTTG AAATCGTTGG TATccgtGAc GAcATCCAAA AAGCtGTTGT TA 212 S. anginosusGGTACTGT TaaaGTCAAt GAtGAAGTTG AAATtGTTGG TATTcgtGAc GAaATCCAAA AAGCAGTTGT TA 213 S. cricetusGGTACTGT TaagGTCAAt GACGAAGTTG AAATCGTTGG TATcAAgGAc GAaATCCAAA AAGCgGTTGT TA 214 S. cristatusGGTACTGT TCGTGTCAAC GAtGAAaTcG AAATCGTTGG TATcAAAGAA GAaATCCAAA AAGCAGTTGT TA 215 S. downeiGGTACTGT TaagGTCAAC GACGAAGTTG AAATCGTTGG TATcAAgGAc GAaATCCAAA AAGCAGTTGT TA 216 S. dysgalactiaeGGTACTGT TCGTGTCAAC GACGAAaTcG AAATCGTTGG TATcAAAGAA GAaActaAAA AAGCtGTTGT TA 217 S. equi equiGGTACTGT TCGTGTtAAC GACGAAaTcG AAATCGTTGG TATcAgAGAc GAgATCaAAA AAGCAGTTGT TA 218 S. ferusGGTACTGT aaGaGTCAAC GAtGAAGTTG AAATCGTTGG TATcAAAGAc GAaATCactA AAGCAGTTGT TA 219 S. gordoniiGGTAtcGT TaaaGTCAAt GACGAAaTcG AAATCGTTGG TATcAAAGAA GAaATCCAAA AAGCAGTTGT TA 220 S. macacaeGGTACTGT TaagGTtAAt GAtGAAGTTG AAATCGTTGG TATTcgtGAc GATATtCAAA AAGCAGTTGT TA 222 S. gordoniiGGTAtcGT TaaaGTCAAC GACGAAaTcG AAATCGTTGG TATcAAAGAA GAaActCAAA AAGCAGTTGT TA 223 S. mutansGGTACTGT TaaaGTtAAC GAtGAAGTTG AAATCGTTGG TATccgtGAt GAcATtCAAA AAGCtGTTGT TA 224 S. oralisGGTACTGT TCGTGTCAAC GACGAAaTcG AAATCGTTGG TATcAAAGAA GAaActCAAA AAGCAGTTGT TA2398 P33170 S. parasanguinisGGTgtTGT TCGTGTCAAt GAtGAAaTcG AAATCGTTGG TATcAAAGAA GAaATCCAAA AAGCAGTTGT TA 225 S. pneumoniaeGGTAtcGT TaaaGTCAAC GACGAAaTcG AAATCGTTGG TATcAAAGAA GAaActCAAA AAGCAGTTGT TA2399 S. pyogenesGGTACTGT TCGTGTCAAC GACGAAaTcG AAATCGTTGG TATcAAAGAA GAaActaAAA AAGCtGTTGT TA2400 Genome project S. rattiGGTACTGT TaaaGTCAAt GACGAAGTTG AAATCGTTGG TATccgtGAt GAcATCCAAA AAGCtGTTGT TA 226 S. salivariusGGTgtTGT TCGTGTCAAt GACGAAGTTG AAATCGTTGG TcTTAAAGAA GAcATCCAAA AAGCAGTTGT TA2401 S. sanguinisGGTAtcGT TaaaGTCAAC GACGAAaTcG AAATCGTTGG TATcAAAGAA GAaATCCAAA AAGCAGTTGT TA 227 S. sobrinusGGTACTGT TaagGTtAAC GACGAAGTTG AAATCGTTGG TATccgtGAc GATATCCAAA AAGCAGTTGT TA 228 S. suisGGTACTGT TCGTGTCAAC GACGAAaTcG AAATCGTTGG TcTTcAAGAA GAaAaatctA AAGCAGTTGT TA 229 S. uberisGGTACTGT TCGTGTCAAC GACGAAaTTG AAATCGTTGG TATcAAAGAA GAaActaAAA AAGCAGTTGT TA 230 S. vestibularisGGTgtTGT TCGTGTtAAt GACGAAGTTG AAATCGTTGG TcTTAAAGAA GAaATCCAAA AAGCAGTTGT TA 231 Selected sequences for    ACTGT TCGTGTCAAC GACGAAGTTG AAA  582species-specific                                   CGTTGG TATTAAAGAA GATATCCAAA AAGCAGTTG 583 hybridization probes^(b) The sequence numbering refers to theStreptococcus agalactiae tuf gene fragment (SEQ ID NO. 209). Nucleotidesin capitals are identical to the selected sequences or match thosesequences. Mismatches are indicated by lower-case letters. Dots indicategaps in the sequences displayed. ^(b)These sequences are thereverse-complement of the selected probes.

TABLE 48 Strategy for the selection of Streptococcus agalactiae-specificamplification primers from atpD sequences.39                                          80   203                             234   368                             399SEQ ID NO.: S. agalactiaeTT GATTGTCTAT AAAAATGGCG ATAAGTCACA AAAAGTAGTA...TAAGGATA CTTTGGGTCG TGTCTTCAAC GTTC...CTT ATTAGCACCT TACTTAAAAG GTGGTAAAG 380 S. agalactiaeTT GATTGTCTAT AAAAATGGCG ATAAGTCACA AAAAGTAGTA...TAAGGATA CTTTGGGTCG TGTCTTCAAC GTTC...CTT ATTAGCACCT TACTTAAAAG GTGGTAAAG 379 S. agalactiaeTT GATTGTCTAT AAAAATGGCG ATAAGTCACA AAAAGTAGTA...TAAGGATA CTTTGGGTCG TGTCTTCAAC GTTC...CTT ATTAGCACCT TACTTAAAAG GTGGTAAAG 381 S. agalactiaeTT GATTGTCTAT AAAAATGGCG ATAAGTCACA AAAAGTAGTA...TAAGGATA CTTTGGGTCG TGTCTTCAAC GTTC...CTT ATTAGCACCT TACTTAAAAG GTGGTAAAG 382 S. agalactiaeTT GATTGTCTAT AAAAATGGCG ATAAGTCACA AAAAGTAGTA...TAAGGATA CTTTGGGTCG TGTCTTCAAC GTTC...CTT ATTAGCACCT TACTTAAAAG GTGGTAAAG 383 S. BovisTT GATTGTtTAT AAAgATGGCG ATAAGTCtCA AAAAaTcGTg...TAAaGAaA CTTTGGGTCG TGTgTTtAAt GTTC...CcT tcTtGCcCCT TACcTAAAAG GTGGTAAAG2402 S. salivariusTT GgTcGTtTAN ActgATGaac AaAAGTCtaA AcgtaTcGTg...TAAaGATA CccTtGGaCG TGTCTTCAAC GTTC...CTT gcTAGCcCCT TACcTtAAgG GTGGTAAAG 387 S. pneumoniaecT tgTcGTCTAc AAAAATGaCG AaAgaaaAac AAAAaTcGTc...TAAaGAaA CTTTGGGaCG TGTCTTCAAC GTTt...CcT tcTtGCcCCT TACcTtAAAG GTGGTAAAG2403 S. pyogenesTT GATTGTtTAT AAAgATaGtG ATAAaaagCA AAAAaTcGTc...TAAaGAaA CTTTGGGaCG cGTCTTtAAt GTaC...CcT tcTtGCcCCT TACcTtAAAG GTGGTAAAG2404 S. anginosuscT tgTaGTCTAT AAAAATGaCG AaAAtaaAtc AAAAaTcGTc...gAAaGAaA CacTtGGTCG cGTCTTtAAC GTTt...CcT tTTAGCcCCc TACcTcAAAG GTGGgAAAG 386 S. sanguiniscT tgTaGTCTAT AAAAATGatG AgAAaaaAtc AAAAaTcGTc...aAAGGAaA CTcTaGGcCG gGTgTTCAAt GTTt...CcT gcTAGCACCT TAtcTgAAAG GTGGgAAAG2405 S. mutansTT GgTcGTtTAT AAAgATGGCG AcAAGTCtCA AAgAaTtGTt...aAAaGAaA CacTaGGTCG TGTCTTtAAt GTTC...CcT tcTtGCcCCT TAtcTtAAAG GTGGTAAAG2406 B. anthracisgT aAaacagagc AAcgAaaaCG gaAcaagcat tAActTAacA...TgAtGcaA CacTtGGTCG TGTaTTtAAC GTat...CTT AcTtGCtCCT TACaTtAAgG GTGGTAAga 247 B. cereusgT aAaacaaagc AAcgAaaaCG g...aagcat gAActTAacA...TgAtGcaA CacTtGGaCG TGTaTTCAAC GTat...CTT AcTtGCtCCT TACaTtAAgG GTGGTAAga 248 E. faeciumTT agTTGTtTAT AAAAATGaCG AaAAtaaAtc AAAAGTtGTt...TAAaGAaA CaTTaGGTCG cGTaTTCAAC GTaC...tTT gcTtGCcCCa TAtTTAAAAG GTGGgAAAG 292 E. gallinarumTT GATcGTtTAc AAAAAaGaCG AgAAaaaAac AAAAGTAGTA...aAcaGATA CTcTaGGcCG aGTaTTtAAt GTaC...tTT ATTAGCtCCT TACTTAAAAG GTGGTAAAG 293 E. faecalisTT agTcGTtTAN AAAAATGGCG AagcaaaACA AAAAGTAGTA...TAAaGATA CaTTaGGTCG TGTgTTtAAC GTTt...CTT ATTAGCACCT TAtcTAAAAG GTGGTAAAG 291 E. coliTa cgaTGctctT gAggtgcaaa ATggtaatgA gcgtcTgGTg...TAAaGcgA CTcTGGGcCG TaTCaTgAAC GTaC...CcT gaTgtgtCCg TtCgctAAgG GcGGTAAAG2407 L. monocytogenesTa tAaatctgAT gcAgAaGaaG caccaaCtag ccAAcTtact...TAcaGtaA CTcTtGGTCG TGTaTTtAAt GTat...CTT gcTAGCtCCT TACTTAAAAG GTGGTAAAa 324 S. aureusgT tATTGatgtg cctAAaGaaG AaggtaCAat AcAAcTAacA...TgAtGAaA CaTTaGGTCG TGTaTTtAAt GTaC...tTT AcTAGCACCT TAtaTtAAAG GTGGTAAAa 366 S. epidermidisca cATcGaagtT cctAAaGaaG ATggagCgCt tcAAtTAacA...TgAcGtaA CTcTaGGaaG aGTgTTtAAC GTaC...CTT ATTAGCACCT TACaTAAAAG GTGGTAAAa 370 Selected sequences        ATTGTCTAT AAAAATGGCG ATAAGTC  627for species-specific                   AAAATGGCG ATAAGTCACA AAAAGTA  628primer Selected sequences                                                    GGATA CTTTGGGTCG TGTCTTCAAC G 625 for species-specific                                                                                              ATTAGCACCT TACTTAAAAG GTGGTA 626 primers^(g) The sequence numbering refers to the Streptococcusagalactiae tuf gene fragment (SEQ ID NO. 380). Nucleotides in capitalsare identical to the selected sequences or match those sequences.Mismatches are indicated by lower-case letters. Dots indicate gaps inthe sequences displayed. ^(a,d,e,f)These sequences were obtained fromGenbank and have accession #: a = AB009314, d = AF001955, e = U31170,and f = V00311. ^(b,c)These sequences were obtained from genomesequencing projects. ^(g)These sequences are the reverse-complement ofthe selected primers.

TABLE 49Strategy for the selection of Candida albicans/dubliniensis-specificamplification primers, Candida albicans-specific hybridization probe andCandida dubliniensis-specific hybridization probe from tuf sequences.SEQ ID Accession337                             368   403                      428   460                              491NO.: #: C. albicansCGTC AAGAAGGTTG GTTACAACCC AAAGACTG...CAACATGA TTGAACCATC CACCAACT...C AAATCCGGTA AAGTTACTGG TAAGACCTTG T 624 — C. albicansCGTC AAGAAGGTTG GTTACAACCC AAAGACTG...CAACATGA TTGAACCATC CACCAACT...C AAATCCGGTA AAGTTACTGG TAAGACCTTG T 409 — C. albicansCGTC AAGAAGGTTG GTTACAACCC AAAGACTG...CAACATGA TTGAACCATC CACCAACT...C AAATCCGGTA AAGTTACTGG TAAGACCTTG T 410 — C. albicansCGTC AAGAAGGTTG GTTACAACCC AAAGACTG...CAACATGA TTGAACCATC CACCAACT...C AAATCCGGTA AAGTTACTGG TAAGACCTTG T 407 — C. albicansCGTC AAGAAGGTTG GTTACAACCC AAAGACTG...CAACATGA TTGAACCATC CACCAACT...C AAATCCGGTA AAGTTACTGG TAAGACCTTG T 408 — C. dubliniensisCGTC AAGAAGGTTG GTTACAACCC AAAGACTG...CAACATGA TTGAAgCtTC CACCAACT...C AAATCCGGTA AgGTTACTGG TAAGACCTTG T 412 — C. dubliniensisCGTC AAGAAGGTTG GTTACAACCC AAAGACTG...CAACATGA TTGAAgCtTC CACCAACT...C AAATCCGGTA AgGTTACTGG TAAGACCTTG T 414 — C. dubliniensisCGTC AAGAAGGTTG GTTACAACCC AAAGACTG...CAACATGA TTGAAgCtTC CACCAACT...C AAATCCGGTA AgGTTACTGG TAAGACCTTG T 415 — C. glabrataCATC AAGAAGGTcG GTTACAACCC AAAGACTG...CAACATGA TTGAAgCcaC CACCAACG...C AAggCtGGTg tcGTcAagGG TAAGACCTTG T 417 — C. guilliermondiiCGTC AAGAAGGTTG GTTACAACCC tAAGACTG...CAACATGA TTGAggCtTC tACCAACT...C AAggCtGGTA AgtccACcGG TAAGACtTTG T 418 — C. kefyrCATC AAGAAGGTcG GTTACAACCC AAAGACTG...CAACATGA TTGAAgCcaC CACCAACG...C AAggCtGGTA ccGTcAagGG TAAGACCTTG T 421 — C. kruseiCATC AAGAAGGTTG GTTACAACCC AAAGACTG...CAACATGA TTGAAgCATC CACCAACT...C AAggCaGGTg ttGTTAagGG TAAGACCTTA T 422 — C. lusitaniaeCGTC AAGAAGGTTG GTTACAACCC tAAGACTG...CAACATGA TTGAgCCATC YACCAACT...C AAgTCYGGTA AgtccACcGG TAAGACCTTG T 424 — C. neoformansCATC AAGAAGGTTG GTTACAACCC cAAGgCTG...CAACATGt TgGAggagaC CACCAAGT...C AAgTCtGGTg tttccAagGG TAAGACCcTC C 623 — C. parapsilosisCGTC AAGAAGGTTG GTTACAACCC tAAagCTG...CAAtATGA TTGAACCATC aACCAACT...T AAAgCtGGTA AgGTTACcGG TAAGACCTTG T 426 — C. tropicalisCGTC AAGAAGGTTG GTTACAACCC tAAGgCTG...CAACATGA TTGAAgCtTC tACCAACT...C AAggCtGGTA AgGTTACcGG TAAGACtTTG T 429 — A. fumigatusCATC AAGAAGGTcG GcTACAACCC cAAGgCCG...CAACATGc TTGAgCCcTC CtCCAACT...C AAggCCGGcA AgGTcACTGG TAAGACCcTC A 404 — HumanCATt AAGAAaaTTG GcTACAACCC cgAcACAG...CAACATGc TgGAgCCAag tgCtAACA...T AAggatGGcA AtGccAgTGG aAccACgcTG C2408 X03558 P. anomalaTATC AAGAAaGTTG GTTACAACCC AAAaACTG...TAACATGA TTGAACCATC aWCtAACT...C AAAgCtGGTg AAGcTAaaGG TAAaACtTTA T 447 — S. cerevisiaeTATC AAGAAGGTTG GTTACAACCC AAAGACTG...CAACATGA TTGAAgCtaC CACCAACG...C AAggCCGGTg tcGTcAagGG TAAGACtTTG T 622 — S. pombeCATC AAGAAGGTcG GTTtCAACCC cAAGACCG...TAACATGA TTGAgCCcaC CACCAACA...C AAggCtGGTg tcGTcAagGG TAAGACtcTT T2409 U42189 Selected sequence    C AAGAAGGTTG GTTACAACCC AAAGAfor species-specific     amplification primer^(a) Selected sequence                                                                        ATCCGGTA AAGTTACTGG TAAGACCTfor species-specific amplification primer^(a,b) Selected sequences                                         CATGA TTGAACCATC CACCA (C. albicans) 577 for species-specific                                         CATGA TTGAAGCTTC CACCA (C. dubliniensis) 578 hybridization probes The sequence numbering refers to the Candidaalbicans tuf gene fragment (SEQ ID NO. 408). Nucleotides in capitals areidentical to the selected sequences or match those sequences. Mismatchesfor SEQ ID NO. 577 are indicated by lower-case letters. Mismatches forSEQ ID NO. 578 are indicated by underlined nucleotides. Dots indicategaps in the sequences displayed. “R” “Y” “M” “K” “W” and “S” designatenucleotide positions which are degenerated. “R” stands for A or G; “Y”stands for C or T; “M” stands for A or C; “K” stands for G or T; “W”stands for A or T; “S” stands for C or G. “I” stands for inosine whichis a nucleotide analog that can bind to any of the four nucleotides A,C, G or T. ^(a) C. albicans primers have been described in a previouspatent (publication WO98/20157, SEQ ID NOs. 11-12) ^(b)This sequence isthe reverse-complement of the selected primer.

TABLE 50 Strategy for the selection of Staphylococcus-specificamplification primers from tuf sequences.310                            340   652                            682SEQ ID NO.: Accession #: S. aureusA CAGGCCGTGT TGAACGTGGT CAAATCAAAG...CACTTACCA GAAGGTACTG AAATGGTAAT GC 179 — S. aureus A CAGGCCGTGT TGAACGTGGT CAAATCAAAG...CACTTACC~~~~~~~~~~~ ~~~~~~~~~~ GC  176 — S. aureusA CAGGCCGTGT TGAACGTGGT CAAATCAAAG...CACTTACCA GAAGGTMCTG AAATGGTAAT GC 177 — S. aureus aureusA CAGGCCGTGT TGAACGTGGT CAAATCAAAG...CACTTACCA GAAGGTACTG AAATGGTAAT GC 180 — S. auricularisA CAGGCCGTGT TGAACGTGGT CAAATCAAAG...ActTTACCA GAAGGTACaG AAATGGTAAT GC 181 — S. capitis capitisA CAGGCCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA GAAGGTACTG AAATGGTTAT GC 182 — M. caseolyticusA CTGGaCGTGT TGAgCGTGGa CAAgTtAAAG...AACTTACCA GAAGGTACTG AAATGGTAAT GC 183 — S. cohniiA CAGGgCGTGT TGAACGTGGT CAAATCAAAG...ActTTACCA GAAGGTACTG AAATGGTTAT GC 184 — S. epidermidis A CAGGCCGTGT TGAACGTGGT CAAATCAAAG...~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~ ~~  185 — S. epidermidisA CAGGCCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA GAAGGTACaG AAATGGTTAT GC  141^(a) — S. haemolyticusA CAGGCCGTGT TGAACGTGGg CAAATCAAAG...AACTTACCA GAAGGTACTG AAATGGTTAT GC 186 — S. haemolyticusA CAGGtCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA GAAG~~~~~~ ~~~~~~~~~~ ~~ 188 — S. haemolyticusA CAGGCCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA GAAGGTACTG AAATGG~~~~ ~~ 189 — S. hominis hominisA CAGGCCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA GAAGGTACTG AAATGGTAAT GC 191 — S. hominisA CAGGCCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA GAAGGTACTG AAATGGTAAT GC 193 — S. hominisA CAGGCCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA GAAGG~~~~~ ~~~~~~~~~~ ~~ 194 — S. hominisA CAGGCCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA GAAGGTACTG AAATGGTAAT GC 195 — S. hominisA CAGGCCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA GAAGGTACTG AAATGGTAAT GC 196 — S. lugdunensisA CAGGCCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA GAAGGTACaG AAATGGTTAT GC 197 — S. saprophyticus A CAGGCCGTGT TGAACGTGGT CAAATCAAAG...~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~ ~~  198 — S. saprophyticusA CAGGCCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA GAAGGTACTG AAATGGTTAT GC 199 — S. saprophyticusA CAGGCCGTGT TGAACGTGGT CAAATCAAAG...AACTTACCA GAAGGTACTG AAATGGTTAT GC 200 — S. sciuri sciuriA CAGGCCGTGT TGAACGTGGT CAAATCACTG...AACTTACCA GAAGGTACTG AAATGGTTAT GC 201 — S. warneriA CAGGCCGTGT TGAACGTGGT CAAATCAAAG...CAaTTACCA GAAGGTACTG ~~~~~~~~~~ ~~ 187 — S. warneri A CAGGCCGTGT TGAACGTGGT CAAATCAAAG...~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~ ~~  192 — S. warneriA CAGGCCGTGT TGAACGTGGT CAAATCAAAG...CAaTTACCA GAAGGTACTG AAATGGTTAT GC 202 — B. subtilisA CTGGCCGTGT aGAACGcGGa CAAgTtAAAG...CAtcTtCCA GAAGGcgtaG AAATGGTTAT GC2410 Z99104 E. coliA CCGGtCGTGT aGAACGcGGT atcATCAAAG...GAacTgCCg GAAGGcgtaG AgATGGTAAT GC  78 — L. monocytogenesA CTGGaCGTGT TGAACGTGGa CAAgTtAAAG...AcacTtCCA GAAGGTACTG AAATGGTAAY GC2411 — Selected sequence      GGCCGTGT TGAACGTGGT CAAATCA  553for genus-specific primer  Selected sequences                                         TTACCA GAAGGTACTG AAATGGTIA  575for genus-specific primers^(b)                                        TTACCA GAAGGTACTG AAATGGTWA  707The sequence numbering refers to the Staphylococcus aureus tuf genefragment (SEQ ID NO. 179). Nucleotides in capitals are identical to theselected sequences or match those sequences. Mismatches are indicated bylower-case letters. “~” indicate incomplete sequence data. Dots indicategaps in the sequences displayed. “R” “Y” “M” “K” “W” and “S” designatenucleotide positions which are degenerated. “R” stands for A or G; “Y”stands for C or T; “M” stands for A or C; “K” stands for G or T; “W”stands for A or T; “S” stands for C or G. “I” stands for inosine whichis a nucleotide analog that can bind to any of the four nucleotides A,C, G or T. ^(b)These sequences are the reverse-complement of theselected primers.

TABLE 51 Strategy for the selection of theStaphylococcus-specific hybridization probe from tuf sequences.400                       425 SEQ ID NO.: Accession #: S. aureusG TTGAAATGTT CCGTAAATTA TTAGA  179 — S. aureusG TTGAAATGTT CCGTAAATTA TTAGA  176 — S. aureusG TTGAAATGTT CCGTAAATTA TTAGA  177 — S. aureusG TTGAAATGTT CCGTAAATTA TTAGA  178 — S. aureus aureusG TTGAAATGTT CCGTAAATTA TTAGA  180 — S. auricularisG TAGAAATGTT CCGTAAATTA TTAGA  181 — S. capitis capitisG TAGAAATGTT CCGTAAATTA TTAGA  182 — M. caseolyticusG TAGAAATGTT CCGTAAATTA TTAGA  183 — S. cohniiG TAGAAATGTT CCGTAAATTA TTAGA  184 — S. epidermidisG TAGAAATGTT CCGTAAATTA TTAGA  185 — S. haemolyticusG TAGAAATGTT CCGTAAATTA TTAGA  186 — S. haemolyticusG TAGAAATGTT CCGTAAATTA TTAGA  189 — S. haemolyticusG TAGAAATGTT CCGTAAATTA TTAGA  190 — S. haemolyticusG TAGAAATGTT CCGTAAATTA TTAGA  188 — S. hominisG TAGAAATGTT CCGTAAATTA TTAGA  196 — S. hominisG TAGAAATGTT CCGTAAATTA TTAGA  194 — S. hominis hominisG TAGAAATGTT CCGTAAATTA TTAGA  191 — S. hominisG TAGAAATGTT CCGTAAATTA TTAGA  193 — S. hominisG TAGAAATGTT CCGTAAATTA TTAGA  195 — S. lugdunensisG TAGAAATGTT CCGTAAATTA TTAGA  197 — S. saprophyticusG TAGAAATGTT CCGTAAATTA TTAGA  198 — S. saprophyticusG TAGAAATGTT CCGTAAATTA TTAGA  200 — S. saprophyticusG TAGAAATGTT CCGTAAATTA TTAGA  199 — S. sciuri sciuriG TTGAAATGTT CCGTAAATTA TTAGA  201 — S. warneriG TAGAAATGTT CCGTAAgTTA TTAGA  187 — S. warneriG TAGAAATGTT CCGTAAgTTA TTAGA  192 — S. warneriG TAGAAATGTT CCGTAAgTTA TTAGA  202 — S. warneriG TAGAAATGTT CCGTAAgTTA TTAGA  203 — B. subtilisG TTGAAATGTT CCGTAAgcTt cTTGA 2412 Z99104 E. coliG TTGAAATGTT CCGcAAAcTg cTGGA   78 — L. monocytogenesG TAGAAATGTT CCGTAAATTA cTAGA 2413 — Selected sequence for    GAAATGTT CCGTAAATTA TT  605 genus-specific hybridization probe Thesequence numbering refers to the Staphylococcus aureus tuf gene fragment(SEQ ID NO. 179). Nucleotides in capitals are identical to the selectedsequence or match that sequence. Mismatches are indicated by lower-caseletters.

TABLE 52Strategy for the selection of Staphylococcus saprophyticus-specific and of Staphylococcus haemolyticus-specific hybridization probes from tuf sequences.SEQ ID 339                                            383 NO.: S. aureusAG TtGGTGAAGA AgTtGAAATC ATcGGTtTaC ATGACACaTC TAA  179 S. aureusAG TtGGTGAAGA AgTtGAAATC ATeGGTtTaC ATGACACaTC TAA  176 S. aureusAG TtGGTGAAGA AgTtGAAATC ATeGGTtTaC ATGACACaTC TAA  177 S. aureusAG TtGGTGAAGA AgTtGAAATC ATeGGTtTaC ATGACACaTC TAA  178 S. aureus aureusAG TtGGTGAAGA AgTtGAAATC ATcGGTtTaC ATGACACaTC TAA  180 S. auricularisAG TCGGTGAAGA AgTtGAAATC ATcGGTATga AaGACggTTC AAA  181S. capitis capitis AG TtGGTGAAGA AgTtGAAATC ATcGGTATCC AcGAaACTTC TAA 182 M. caseolyticus AG TtGGTGAAGA AgTtGAAATC ATTGGTtTaa cTGAagaacC AAA 183 S. cohnii AG TCGGTGAAGA AgTtGAAATC ATcGGTATgC AaGAagaTTC CAA  184S. epidermidis AG TtGGTGAAGA AgTtGAAATC ATcGGTATgC AcGAaACTTC TAA  185S. haemolyticus AG TtGGTGAAGA AgTtGAAATC ATTGGTATCC ATGACACTTC TAA  186S. haemolyticus AG TtGGTGAAGA AgTtGAAATC ATTGGTATCC ATGACACTTC TAA  189S. haemolyticus AG TtGGTGAAGA AgTtGAAATC ATTGGTATCC ATGACACTTC TAA  190S. haemolyticus AG TtGGTGAAGA AgTtGAAATt ATTGGTATCa AaGAaACTTC TAA  188S. hominis AG TtGGTGAAGA AgTtGAAATt ATTGGTATCa AaGAaACTTC TAA  194S. hominis hominis AG TtGGTGAAGA AgTtGAAATt ATTGGTATCa AaGAaACTTC TAA 191 S. hominis AG TtGGTGAAGA AgTtGAAATt ATTGGTATCa AaGAaACTTC TAA  193S. hominis AG TtGGTGAAGA AgTtGAAATt ATTGGTATCa AaGAaACTTC TAA  195S. hominis AG TtGGTGAAGA AgTtGAAATt ATTGGTATCa AaGAtACTTC TAA  196S. lugdunensis AG TCGGTGAAGA AgTtGAAATt ATTGGTATCC AcGAtACTaC TAA  197S. saprophyticus AG TCGGTGAAGA AATCGAAATC ATcGGTATgC AaGAagaaTC CAA  198S. saprophyticus AG TCGGTGAAGA AATCGAAATC ATcGGTATgC AaGAagaaTC CAA  200S. saprophyticus AG TCGGTGAAGA AATCGAAATC ATcGGTATgC AaGAagaaTC CAA  199S. sciuri sciuri TG TtGGTGAAGA AgTtGAAATC ATcGGTtTaa cTGAagaaTC TAA  201S. warneri AG TtGGTGAAGA AgTtGAAATC ATcGGTtTaC ATGACACTTC TAA  187S. warneri AG TtGGTGAAGA AgTtGAAATC ATeGGTtTaC ATGACACTTC TAA  192S. warneri AG TtGGTGAAGA AgTtGAAATC ATcGGTtTaC ATGACACTTC TAA  202S. warneri AG TtGGTGAAGA AgTtGAAATC ATcGGTtTaC ATGACACTTC TAA  203B. subtilis AG TCGGTGAcGA AgTtGAAATC ATcGGTcTtC AaGAagagag AAA  2414^(a)E. coli AG TtGGTGAAGA AgTtGAAATC gTTGGTATCa AaGAgACTca GAA   78L. monocytogenes AG TtGGTGAcGA AgTaGAAgTt ATcGGTATCg AaGAagaaag AAA 2415Selected sequences for CGGTGAAGA AATCGAAATC A (S. saprophyticus)  599species-specific    (S. haemolyticus) ATTGGTATCC ATGACACTTC  594hybridization probes The sequence numbering refers to the Staphylococcusaureus tuf gene fragment (SEQ ID NO. 179). Nucleotides in capitals areidentical to the selected sequences or match those sequences. Mismatchesare indicated by lower-case letters. ^(a)This sequence was obtained fromGenbank accession #Z99104.

TABLE 53Strategy for the selection of Staphylococcus aureus-specific and ofStaphylococcus epidermidis-specific hybridization probes from tuf sequences.SEQ ID 521                      547   592                      617 NO.:S. aureus TACACCACA TACTGAATTC AAAGCAG...TTCTTCtCa AACTATCGtC CACAATT 179 S. aureus TACACCACA TACTGAATTC AAAGCAG...TTCTTCtC~ ~~~~~~~~~~~~~~~~~  178 S. aureusTACACCACA TACTGAATTC AAAGCAG...TTCTTCtCa AACTATCGtC CACAATT  176S. aureus TACACCACA TACTGAATTC AAAGCAG...TTCTTCtCa AACTATCGtC CACAATT 177 S. aureus aureusTACACCACA TACTGAATTC AAAGCAG...TTCTTCtCa AACTATCGtC CACAATT  180S. auricularisTACACCACA cACTaAATTC ActGCAG...TTCTTCtCT AACTAcCGtC CACAATT  181S. capitis capitisCACACCACA cACTaAATTC AAAGCGG...TTCTTCAgT AACTAcCGCC CACAATT  182M. caseolyticusTACtCCACA TACTaAATTC AAAGCTG...TTCTTCACT AACTAcCGCC CtCAGTT  183S. cohnii TACACCACA cACaaAcTTt AAAGCGG...TTCTTCAgT AACTATCGCC CACAATT 184 S. epidermidisTACACCACA cACaaAATTC AAAGCTG...TTCTTCACT AACTATCGCC CACAATT  185S. haemolyticusCACACCtCA cACaaAATTt AAAGCAG...TTCTTCACa AACTATCGtC CACAATT  186S. haemolyticusCACACCtCA cACaaAATTt AAAGCAG...TTCTTCACa AACTATCGtC CACAATT  189S. haemolyticusCACACCtCA cACaaAATTt AAAGCAG...TTCTTCACa AACTATCGtC CACAATT  190S. haemolyticusTACACCtCA cACaaAATTC AAAGCAG...TTCTTCACT AACTATCGtC CACAATT  188S. hominis CACACCtCA cACaaAATTC AAAGCAG...TTCTTCACT AACTATCGtC CACAATT 195 S. hominisTACACCtCA cACaaAATTC AAAGCAG...TTCTTCACT AACTATCGtC CACAATT  196S. hominis hominisTACACCtCA cACaaAATTC AAAGCAG...TTCTTCtCT AACTATCGtC CACAATT  191S. hominis TACACCtCA cACaaAATTC AAAGCAG...TTCTTCtCT AACTATCGtC CACAATT 193 S. hominisTACACCtCA cACaaAATTC AAAGCAG...TTCTTCtCT AACTATCGtC CACAATT  194S. lugdunensisTACACCtCA cACTaAATTt AAAGCTG...TTCTTCtCa AACTAcCGCC CACAATT  197S. saprophyticusTACACCACA TACaaAATTC AAAGCGG...TTCTTCACT AACTAcCGCC CACAATT  198S. saprophyticusTACACCACA TACaaAATTC AAAGCGG...TTCTTCACT AACTAcCGCC CACAATT  199S. saprophyticusTACACCACA TACaaAATTC AAAGCGG...TTCTTCACT AACTAcCGCC CACAATT  200S. sciuri sciuriCACACCtCA cACTaAATTC AAAGCTG...TTCTTCACa AACTAcCGCC CACAATT  201S. warneri TACACCACA TACaaAATTC AAAGCGG...~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~ 192 S. warneriTACACCACA TACaaAATTC AAAGCGG...TTCTTCAgT AACTAcCGCC CACAATT  187S. warneri TACACCACA TACaaAATTC AAAGCGG...TTCTTCAgT AACTAcCGCC CACAATT 202 S. warneriTACACCACA TACaaAATTC AAAGCGG...TTCTTCAgT AACTAcCGCC CACAATT  203B. subtilis CACtCCACA cAgCaAATTC AAAGCTG...TTCTTCtCT AACTAcCGtC CtCAGTT2416 E. coli CAAgCCgCA cACcaAgTTC gAAtCTG...TTCTTChaa ggCTAcCGtC CgCAGTT  78 L. monocytogenesTACtCCACA cACTaAcTTC AAAGCTG...TTCTTCAac AACTAcCGCC CACAATT 2417Selected sequences    ACCACA TACTGAATTC AAAG (S. aureus)  585for species-specific                 (S. epidermidis) TTCACT AACTATCGCC CACA  593hybridization probes The sequence numbering refers to the Staphylococcusaureus tuf gene fragment (SEQ ID NO. 179). Nucleotides in capitals areidentical to the selected sequences or match those sequences. Mismatchesare indicated by lower-case letters. “~” indicate incomplete sequencedata. Dots indicate gaps in the sequences displayed. ^(a)This sequencewas obtained from Genbank accession #Z99104.

TABLE 54 Strategy for the selection of the Staphylococcus hominis-specific hybridization probe from tuf sequences.358                       383 SEQ ID NO.: S. aureusATC ATcGGTtTac AtGAcACaTC TAA  179 S. aureusATC ATcGGTtTac AtGAcACaTC TAA  176 S. aureusATC ATcGGTtTac AtGAcACaTC TAA  177 S. aureusATC ATcGGTtTac AtGAcACaTC TAA  178 S. aureus aureusATC ATcGGTtTac AtGAcACaTC TAA  180 S. auricularisATC ATcGGTATgA AAGAcggTTC AAA  181 S. capitis capitisATC ATcGGTATCc AcGAAACTTC TAA  182 M. caseolyticusATC ATTGGTtTaA ctGAAgaacC AAA  183 S. cohniiATC ATcGGTATgc AAGAAgaTTC CAA  184 S. epidermidisATC ATcGGTATgc AcGAAACTTC TAA  185 S. haemolyticusATC ATTGGTATCc AtGAcACTTC TAA  186 S. haemolyticusATC ATTGGTATCc AtGAcACTTC TAA  189 S. haemolyticusATC ATTGGTATCc AtGAcACTTC TAA  190 S. haemolyticusATT ATTGGTATCA AAGAAACTTC TAA  188 S. hominisATT ATTGGTATCA AAGAtACTTC TAA  196 S. hominisATT ATTGGTATCA AAGAAACTTC TAA  194 S. hominis hominisATT ATTGGTATCA AAGAAACTTC TAA  191 S. hominisATT ATTGGTATCA AAGAAACTTC TAA  193 S. hominisATT ATTGGTATCA AAGAAACTTC TAA  195 S. lugdunensisATT ATTGGTATCc AcGAtACTaC TAA  197 S. saprophyticusATC ATcGGTATgc AAGAAgaaTC CAA  198 S. saprophyticusATC ATcGGTATgc AAGAAgaaTC CAA  200 S. saprophyticusATC ATcGGTATgc AAGAAgaaTC CAA  199 S. sciuri sciuriATC ATcGGTtTaA ctGAAgaaTC TAA  201 S. warneriATC ATcGGTtTac AtGAcACTTC TAA  187 S. warneriATC ATcGGTtTac AtGAcACTTC TAA  192 S. warneriATC ATcGGTtTac AtGAcACTTC TAA  202 S. warneriATC ATcGGTtTac AtGAcACTTC TAA  203 B. subtilisATC ATcGGTcTtc AAGAAgagag AAA  2418^(a) E. coliATC gTTGGTATCA AAGAgACTca GAA   78 L. monocytogenesGTT ATcGGTATCg AAGAAgaaag AAA 2419 Selected sequence for    ATTGGTATCA AAGAAACTTC  597 species-specific hybridization probe Thesequence numbering refers to the Staphylococcus aureus tuf gene fragment(SEQ ID NO. 179). Nucleotides in capitals are identical to the selectedsequences or match those sequences. Mismatches are indicated bylower-case letters. Dots indicate gaps in the sequences displayed.^(a)This sequence was obtained from Genbank accession #Z99104.

TABLE 55 Strategy for the selection of the Enterococcus-specificamplification primers from tuf sequences. SEQ Acces- ID sion270                         298   556                        582 NO.: #: E. aviumTAGAATTAAT GGCTGCTGTT GACGAATAT...TGAA GATATCCAAC GTGGACAAGT ATT 2420 —E. casseliflavusTGGAATTAAT GGCTGCAGTT GACGAATAC...TGAA GACATCCAAC GTGGACAAGT ATT   58 —E. cecorumTAGAATTAAT GGCTGCAGTT GACGAATAC...TGAA GATATCCAAC GTGGtCAAGT ATT   59 —E. disparTAGAATTAAT GGCTGCAGTT GACGAATAT...TGAA GATATCCAAC GTGGtCAAGT ATT   60 —E. duransTTGAATTAAT GGCTGCAGTT GACGAATAT...TGAA GACATCCAAC GTGGACAAGT TTT   61 —E. flavescensTGGAATTAAT GGCTGCAGTT GACGAATAC...TGAA GACATCCAAC GTGGACAAGT ATT   65 —E. faeciumTTGAATTAAT GGCTGCAGTT GACGAATAC...TGAA GACATCCAAC GTGGACAAGT TTT  608 —E. faecalisTAGAATTAAT GGCTGCAGTT GACGAATAT...TGAA GATATCGAAC GTGGACAAGT ATT  607 —E. gallinarumTGGAATTgAT GGCTGCAGTT GACGAATAC...TGAA GACATCCAAC GTGGACAAGT ATT  609 —E. hiraeTTGAATTgAT GGCTGCAGTT GACGAATAT...TGAA GACATCCAAC GTGGACAAGT TTT   67 —E. mundtiiTTGAATTgAT GGCTGCAGTT GACGAATAT...TGAA GACATCCAAC GTGGtCAAGT TTT   68 —E. pseudoaviumTAGAATTAAT GSCTGCTGTT GACGAATAC...TGAA GACATCCAAC GTGGACAAGT ATT   69 —E. raffinosusTAGAATTAAT GGCTGCTGTT GATGAATAC...TGAA GACATCCAAC GTGGACAAGT ATT   70 —E. saccharolyticusTCGAATTAAT GGCTGCAGTT GACGAATAT...TGAA GACATCCAAC GTGGACAAGT ATT   71 —E. solitariusTGGAATTAAT GGaTGCAGTT GATGAcTAC...TGAt GATATCGAAC GTGGtCAAGT ATT   72 —E. coli TGGAAcTggc tGgcttccTg GATtctTAY...TGAA GAaATCGAAC GTGGtCAgGT ACT  78 — B. cepaciaTGAgccTggc cGacGCgcTg GACacgTAC...TGAA GACgTgGAgC GTGGcCAgGT TCT   16 —B. fragilisTGGAAcTgAT GGaaGCTGTT GATactTGG...GAAc GAaATCaAAC GTGGtatgGT TCT 2421M22247 B. subtilisTCGAAcTtAT GGaTGCgGTT GATGAgTAC...TGAA GATATCCAAC GTGGtCAAGT ACT 2422Z99104 C. diphtheriaeTCGAccTcAT GcagGCTtgc KATGAtTCC...CGAA GACgTtGAgC GTGGcCAgGT TGT  662 —C. trachomatisGAGAgcTAAT GcaaGCcGTc GATGAtAAT...GAAc GATgTgGAAa GaGGAatgGT TGT   22 —G. vaginalisAGGAAcTcAT GaagGCTGTT GACGAgTAC...TACc GACgTtGAgC GTGGtCAgGT TGT 2423 —S. aureusTAGAATTART GGaaGCTGTa GATactTAC...TGAA GACgTaCAAC GTGGtCAAGT ATT  179 —S. pneumoniaeTGGAATTgAT GaacaCAGTT GATGAgTAT...TGAt GAaATCGAAC GTGGACAAGT TAT 2424 —A. adiacensTAGAATTAAT GGCTGCTGTT GACGAATAC...TGAA aACATCGAAC GTGGACAAGT TCT 2425 —G. haemolysansTCGAATTAAT GGaaaCAGTT GACGAATAC...TGAA GACATCGAAC GTGGACAAGT TTT   87 —G. morbillorumTCGAATTAAT GGaaaCAGTT GACGAgTAC...TGAA GATATCGAAC GTGGACAAGT TTT   88 —Selected    AATTAAT GGCTGCWGTT GAYGAA 1137 sequence foramplification primer Selected                                                    A GAYATCSAAC GTGGACAAGT 1136sequence for amplification primer^(b) The sequence numbering refers tothe Enterococcus durans tuf gene fragment (SEQ ID NO. 61). Nucleotidesin capitals are identical to the selected sequences or match thosesequences. Mismatches are indicated by lower-case letters. Dots indicategaps in the sequences displayed. “Y” “W” and “S” designate nucleotidepositions which are degenerated. “Y” stands for C or T; “W” stands for Aor T; “S” stands for C or G. “I” stands for inosine which is anucleotide analog that can bind to any of the four nucleotides A, C, Gor T. ^(b)This sequence is the reverse-complement of the selectedprimer.

TABLE 56Strategy for the selection of the Enterococcus faecalis-specific hybridization probe, of the Enterococcus faecium-specific hybridization probe and of the Enterococcus casseliflavus-flavescens-gallinarum group-specific hybridization probe from tuf sequences.395                                                     448...526                    549SEQ ID NO.: Accession #: E. aviumGTTGA ACGTGGacAA GTTCGCGTTG GTGACGAAGT TGAAaTcGTa GGTATcGCT...CATc GGTGCtTTGt TACGTGGTGT2426 — E. casseliflavus GTTGA ACGTGGacAA GTTCGCGTTG GTGACGAAGT TGAAaTcGTT GGTATTGCT...CATT GGTGCATTGC TACGTGGTGT  58 — E. cecorumGTTGA ACGTGGacAA GTaCGtGTTG GTGACGAAGT TGAAaTaGTT GGTATcCAT...CATc GGTGCATTat TACGTGGTGT  59 — E. disparGTTGA ACGTGGacAA GTTCGCGTTG GTGACGAAGT TGAAaTcGTa GGTATcGCT...CATT GGTGCATTat TACGTGGTGT  60 — E. duransGTTGA ACGTGGacAA GTTCGCGTTG GTGACGttGT aGAtaTcGTT GGTATcGCA...CATT GGTGCtTTaC TACGTGGTGT  61 — E. faecalisGTTGA ACGTGGTGAA GTTCGCGTTG GTGACGAAGT TGAAaTcGTT GGTATTAAA...CTTc GGTGCtTTat TACGTGGTGT  62 — E. faeciumGTTGA ACGTGGacAA GTTCGCGTTG GTGACGAAGT TGAAGTTGTT GGTATTGCT...CATT GGTGCtTTaC TACGTGGTGT 608 — E. flavescensGTTGA ACGTGGacAA GTTCGCGTTG GTGACGAAGT TGAAaTcGTT GGTATTGCT...CATT GGTGCATTGC TACGTGGGGT  65 — E. gallinarumGTTGA ACGTGGacAA GTTCGCGTTG GTGATGAAGT aGAAaTcGTT GGTATTGCT...CATT GGTGCATTGC TACGTGGGGT 609 — E. hiraeGTTGA ACGTGGacAA GTTCGCGTTG GTGACGttGT aGAtaTcGTT GGTATcGCA...CATT GGTGCtTTaC TACGTGGTGT  67 — E. mundtiiGTTGA ACGTGGacAA GYTCGtGTTG GTGACGttaT cGAtaTcGTT GGTATcGCA...CATT GGTGCgTTaC TACGTGGTGT  68 — E. pseudoaviumGTTGA ACGTGGacAA GTTCGCGTTG GTGACGAAGT TGAAaTcGTa GGTATCGCT...CATc GGTGCATTat TACGTGGTGT  69 — E. raffinosusGTTGA ACGTGGacAA GTTCGCGTTG GTGACGAAGT TGAAaTcGTa GGTATTGCT...CATT GGTGCATTat TACGTGGTGT  70 — E. saccharolyticusGTTGA ACGTGGacAA GTTCGCGTTG GTGACGttGT aGAAaTcGTT GGTATcGAC...CATc GGTGCtTTat TACGTGGGGT  71 — E. solitariusGTTGA ACGcGGgact aTcaaaGTCG GCGATGAAGT TGAcaTTaTT GGTATTCAT...CATT GGTaCtTTGt TACGTGGTGT  72 — C. diphtheriaeGTTGA gCGTGGctcc cTgaagGTCA ACGAGGAcGT cGAgaTcaTc GGTATcCGC...CTGT GGTctgcTtC TcCGTGGCGT 662 — G. vaginalisGTTGA gCGTGGTaAg cTcCcaATCA ACACCCcAGT TGAgaTcGTT GGTtTgCGC...CACT GGTcttcTtC TcCGcGGTAT 135^(a) — B. cepaciaGTCGA gCGcGGcatc GTgaagGTCG GCGAAGAAaT cGAAaTcGTc GGTATcAAG...CGTT GGTatccTGC TgCGcGGCAC  16 — S. aureusGTTGA ACGTGGTcAA aTcaaaGTTG GTGAAGAAGT TGAAaTcaTc GGTtTaCAT...CATT GGTGCATTat TACGTGGTGT 179 — B. subtilisGTAGA ACGcGGacAA GTTaaaGTCG GTGACGAAGT TGAAaTcaTc GGTcTTCAA...CATT GGTGCccTtC TtCGcGGTGT2427 Z99104 S. pneumoniaeATCGA cCGTGGTatc GTTaaaGTCA ACGACGAAaT cGAAaTcGTT GGTATcAAA...CGTa GGTGtccTtC TtCGTGGTGT2428 — E. coliGTAGA ACGcGGTatc aTcaaaGTTG GTGAAGAAGT TGAAaTcGTT GGTATcAAA...CGTa GGTGttcTGC TgCGTGGTAT  78 — B. fragilisATCGA AacTGGTGtt aTcCatGTAG GTGATGAAaT cGAAaTccTc GGTtTgGGT...CGTa GGTctgTTGC TtCGTGGTGT2429 M22247 C. trachomatisATTGA gCGTGGaatt GTTaaaGTTT CCGATAAAGT TcAgtTgGTc GGTcTTAGA...CGTT GGattgcTcC TcaGaGGTAT  22 — Selected sequences for     GA ACGTGGTGAA GTTCGC (E. faecalis)1174 species-specific or                                   AAGT TGAAGTTGTT GGTATT (E. faecium) 602 group-specific                                                                 T GGTGCATTGC TACGTGG1122 hybridization probes The sequence numbering refers to theEnterococcus faecium tuf gene fragments (SEQ ID NO. 608). Nucleotides incapitals are identical to the selected sequences or match thosesequences. Mismatches are indicated by lower-case letters. Dots indicategaps in the sequences displayed.

TABLE 57 Strategy for the selection of primers for the identification ofplatelets contaminants from tuf sequences. SEQ ID Accession467                          495   689                          717 NO.: #: B. cereusGTA ACTGGTGTaG AGATGTTCCG TAAACT...C AGTTCTACTT CCGTACAACT GACGTAAC    7— B. subtilisGTT ACaGGTGTTG AAATGTTCCG TAAGCT...C AGTTCTACTT CCGTACAACT GACGTAAC 2430Z99104 E. cloacaeTGT ACTGGCGTTG AAATGTTCCG CAAACT...C AGTTCTACTT CCGTACAACT GACGTGAC   54— E. coliTGT ACTGGCGTTG AAATGTTCCG CAAACT...C AGTTCTACTT CCGTACTACT GACGTGAC   78— K. oxytocaTGT ACTGGCGTTG AAATGTTCCG CAAACT...C AGTTCTACTT CCGTACAACT GACGTGAC  100— K. pneumoniaeTGT ACTGGCGTTG AAATGTTCCG CAAACT...C AGTTCTACTT CCGTACTACT GACGTGAC  103— P. aeruginosaTGC ACcGGCGTTG AAATGTTCCG CAAACT...C AGTTCTACTT CCGTACCACK GACGTGAC  153— S. agalactiaeGTT ACTGGTGTTG AAATGTTCCG TAAACA...C AATTCTACTT CCGTACAACT GACGTAAC  209— S. aureusGTT ACaGGTGTTG AAATGTTCCG TAAATT...C AATTCTATTT CCGTACTACT GACGTAAC 2431— S. choleraesuisTGT ACTGGCGTTG AAATGTTCCG CAAACT...C AGTTCTACTT CCGTACTACT GACGTGAC  159— S. epidermidisGTT ACTGGTGTaG AAATGTTCCG TAAATT...C AATTCTATTT CCGTACTACT GACGTAAC  611— S. marcescensTGT ACTGGCGTTG AAATGTTCCG CAAACT...C AGTTCTACTT CCGTACCACT GACGTGAC  168— S. mutansGTT ACTGGTGTTG AAATGTTCCG TAAACA...C AATTCTACTT CCGTACAACT GACGTAAC  224— S. pyogenesGTT ACTGGTGTTG AAATGTTCCG TAAACA...C AATTCTACTT CCGTACAACT GACGTAAC 2432U40453 S. salivariusGTT ACTGGTGTTG AAATGTTCCG TAAACA...C AGTTCTACTT CCGTACAACT GACGTAAC 2433— S. sanguinisGTT ACTGGTGTTG AAATGTTCCG TAAACA...C AGTTCTACTT CCGTACAACT GACGTTAC  227— Y. enterocoliticaTGT ACTGGCGTTG AAATGTTCCG CAAACT...C AGTTCTACTT CCGTACAACT GAtGTAAC  235— Selected sequence      ACTGGYGTTG AIATGTTCCG YAA  636for amplification primer    Selected sequence                                        TTCTAYTT CCGTACIACT GACGT  637for amplification primer^(b)    The sequence numbering refers to the E.coli tuf gene fragment (SEQ ID NO. 78). Nucleotides in capitals areidentical to the selected sequences or match those sequences. Mismatchesare indicated by lower-case letters. Dots indicate gaps in the sequencesdisplayed. “R” “Y” “M” “K” “W” and “S” designate nucleotide positionswhich are degenerated. “R” stands for A or G; “Y” stands for C or T; “M”stands for A or C; “K” stands for G or T; “W” stands for A or T; “S”stands for C or G. “I” stands for inosine which is a nucleotide analogthat can bind to any of the four nucleotides A, C, G or T. ^(b)Thissequence is the reverse-complement of the selected primer.

TABLE 58Strategy for the selection of the universal amplification primers from atpD sequences.616                                        657   781                             812SEQ ID NO. ACCESSION #: C. glutamicumGTGTTCGGTC AGATGGATGA GCCACCAGGA GTCCGTATG CGC...CGTATg CCTTCCGCCG TGGGTTACCA GCCAAC2434 X76875 M. tuberculosisGTATTCGGAC AGATGGACGA GCCGCCGGGC aCCCGTATG CGT...CGGATg CCGTCGGCCG TGGGATACCA GCCCAC2435 Z73419 E. faecalisGTGTTCGGAC AAATGAACGA ACCACCAGGT GCTCGGATG CGG...CGTATg CCTTCTGCCG TTGGTTACCA ACCAAC 291 — S. agalactiaeGTCTTTGGTC AAATGAATGA ACCACCAGGA GCACGTATG CGT...CGTATg CCTTCAGCCG TTGGTTATCA ACCAAC 380 — B. subtilisGTATTCGGAC AAATGAACGA GCCGCCGGGC GCACGTATG CGT...CGTATg CCTTCAGCGG TTGGTTATCA GCCGAC2436 Z28592 L. monocytogenesGTATTCGGTC AAATGAACGA GCCACCAGGT GCGCGTATG CGT...CGTATg CCATCTGCGG TAGGTTACCA ACCAAC 324 — S. aureusGTATTCGGGC AAATGAATGA GCCACCTGGT GCACGTATG CGT...CGTATg CCTTCTGCAG TAGGTTACCA ACCAAC 366 — A. baumanniiGTCTACGGTC AGATGAACGA GCCACCAGGT aaCCGTtTa CGC...CGTATg CCATCTGCGG TAGGTTACCA ACCTAC 243 — N. gonorrhoeaeGTGTATGGCC AAATGAACGA ACCTCCAGGC aaCCGTcTG CGC...CGTATg CCTTCTGCAG TGGGTTACCA ACCGAC2437 Genome project C. freundiiGTATATGGCC AGATGAACGA GCCGCCTGGA aaCCGTcTG CGT...CGTATg CCATCAGCGG TAGGCTACCA GCCGAC 264 — E. cloacaeGTTTACGGCC AGATGAACGA GCCACCAGGA aaCCGTcTG CGC...CGTATg CCTTCAGCGG TAGGTTATCA GCCTAC 284 — E. coliGTGTATGGCC AGATGAACGA GCCGCCGGGA aaCCGTcTG CGC...CGTATg CCTTCAGCGG TAGGTTATCA GCCGAC 669 V00267 S. typhimuriumGTGTATGGCC AGATGAACGA GCCGCCGGGA aaCCGTcTG CGC...CGTATg CCTTCCGCAG TAGGTTACCA GCCGAC 351 — K. pneumoniaeGTGTACGGCC AGATGAACGA GCCGCCGGGA aaCCGTcTG CGC...CGTATg CCTTCAGCGG TAGGTTATCA GCCGAC 317 — S. marcescensGTTTACGGCC AGATGAACGA GCCACCAGGT aaCCGTcTG CGC...CGTATg CCATCCGCGG TAGGTTATCA GCCAAC 357 — Y. enterocoliticaGTTTATGGCC AAATGAATGA GCCACCAGGT aaCCGTcTG CGC...CGTATg CCATCTGCCG TAGGTTACCA GCCAAC 393 — B. cepaciaGTGTACGGCC AGATGAACGA GCCGCCGGGC aaCCGTcTG CGC...CGTATg CCGTCGGCAG TGGGCTATCA GCCGAC2438 X76877 H. influenzaeGTTTATGGTC AAATGAACGA GCCACCAGGT aaCCGTtTa CGT...CGTATg CCATCCGCGG TAGGTTACCA ACCGAC2439 U32730 M. pneumoniaeGTGTTTGGTC AGATGAACGA ACCCCCAGGA GCACGGATG CGG...CGGATg CCATCAGCCG TGGGTTACCA ACCAAC2440 U43738 H. pyloriTGCTATGGGC AAATGAATGA GCCACCAGGT GCAAGGAat CGC...CGTATC CCTTCAGCGG TGGGGTATCA GCCCAC 670 V00267 B. fragilisGTGTTCGGAC AGATGAACGA ACCTCCTGGA GCACGTgct TCA...CGTATg CCTTCTGCGG TAGGTTATCA ACCTAC2441 M22247 Selected sequences for         C ARATGRAYGA RCCICCIGGI GYIMGIATG  562 universal primers   TAYGGIC ARATGAAYGA RCCICCIGGI AA  564 Selected sequences for                                                    ATH CCITCIGCIG TIGGITAYCA RCC 565 universal primers^(a)                                                    ATG CCITCIGCIG TIGGITAYCA RCC 563 The sequence numbering refers to the Escherichia coli atpD genefragment (SEQ ID NO. 669). Nucleotides in capitals are identical to theselected sequences or match those sequences. Mismatches for SEQ ID NOs.562 and 565 are indicated by lower-case letters. Mismatches for SEQ IDNOs. 564 and 563 are indicated by underlined nucleotides. Dots indicategaps in the sequences displayed. “R” “Y” “M” “K” “W” and “S” lettersdesignate nucleotide positions which are degenerated. “R” stands for Aor G; “Y” stands for C or T; “M” stands for A or C; “K” stands for G orT; “W” stands for A or T; “H” stands for A, C or T; “S” stands for C orG. “I” stands for inosine which is a nucleotide analog that can bind toany of the four nucleotides A, C, G or T. ^(a)These sequences are thereverse-complement of the selected primers.

TABLE 59 Specific and ubiquitous primers for nucleicacid amplification (recA sequences). Originating DNA fragment SEQ SEQNucleotide ID NO. Nucleotide sequence ID NO. positionUniversal primers (recA) 919 5′-GGI CCI GAR TCI TMI GGI AAR AC  918^(a)437-459 920^(b) 5′-TCI CCV ATI TCI CCI TCI AIY TC  918^(a) 701-723 9215′-TIY RTI GAY GCI GAR CAI GC  918^(a) 515-534 922^(b)5′-TAR AAY TTI ARI GCI YKI CCI CC  918^(a) 872-894Sequencing primers (recA) 1605 5′-ATY ATY GAA RTI TAY GCI CC 1704^(a)220-239 1606 5′-CCR AAC ATI AYI CCI ACT TTT TC 1704^(a) 628-650Universal primers (rad51) 935 5′-GGI AAR WSI CAR YTI TGY CAY AC  939^(a)568-590 936^(b) 5′-TCI SIY TCI GGI ARR CAI GG  939^(a) 1126-1145Universal primers (dmc1) 937 5′-ATI ACI GAR GYI TTY GGI GAR TT  940^(a)1038-1060 938^(b) 5′-CYI GTI GYI SWI GCR TGI GC  940^(a) 1554-1573^(a)Sequences from databases. ^(b)These sequences are from thecomplementary DNA strand of the sequence of the originating fragmentgiven in the Sequence Listing.

TABLE 60 Specific and ubiquitous primers for nucleicacid amplification (speA sequences). Originating DNA fragment SEQ IDNucleotide SEQ ID NO. Nucleotide sequence NO. positionBacterial species: Streptococcus pyogenes 9945′-TGG ACT AAC AAT CTC GCA AGA GG 993^(a) 60-82  995^(b)5′-ACA TTC TCG TGA GTA ACA GGG T 993^(a) 173-194 9965′-ACA AAT CAT GAA GGG AAT CAT TTA G 993^(a) 400-424  997^(b)5′-CTA ATT CTT GAG CAG TTA CCA TT 993^(a) 504-526 9985′-GGA GGG GTA ACA AAT CAT GAA GG 993^(a) 391-413  997^(b)5′-CTA ATT CTT GAG CAG TTA CCA TT 993^(a) 504-526 ^(a)Sequence fromdatabases. ^(b)These sequences are from the complementary DNA strand ofthe sequence of the originating fragment given in the Sequence Listing.

TABLE 61 First strategy for the selection of Streptococcus pyogenes-specific amplification primers from speA sequences. ACCESSION #57                            85   170                         197SEQ ID NO.: speA X61573CCTT GGgCTAACAA cCTCaCAAGA aGTAT...GTGAtCCT.GT cgtTCAtGAG AATGTAAA 2442speA AF029051 ~~~~GGgCTAACAA cCTCaCAAGA aGTAT...GTGAtCCT.GT cgtTCAtGAG AATGTAAA 2443 speAX61571TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2444speA X61570TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2445speA X61568TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2446speA X61569TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2447speA X61572TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2448speA X61560TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2449speA U40453TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA  993speA X61554TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2450speA X61557TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2451speA X61559TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2452speA X61558TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2453speA X61556TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2454speA X61555TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2455speA X61560TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2456speA X61561TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2457speA X61566TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2458speA X61567TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2459speA X61562TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2460speA X61563TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2461speA X61564TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2462speA X61565TCTT GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2463speA AF055698 ~~~~GGACTAACAA TCTCGCAAGA GGTAT...GTGACCCT.GT TACTCACGAG AATGTGAA 2464 speAX03929^(a)TCTT GGACTAACAA TCTtGCcAaA aGGTA...GTGACCCTGGT TACTCACGAG AATGTGAA 2465Selected sequence for    T GGACTAACAA TCTCGCAAGA GG  994species-specific  primer Selected sequence for                                      ACCCT.GT TACTCACGAG AATGT  995species-specific  primer^(b) The sequence numbering refers to theStreptococcus pyogenes speA gene fragment (SEQ ID NO. 993). Nucleotidesin capitals are identical to the selected sequences or match thosesequences. Mismatches are indicated by lower-case letters. “~” indicateincomplete sequence data. Dots indicate gaps in the sequences displayed.^(a)The extra G nucleotide introducing a gap in the sequence is probablya sequencing error. ^(b)This sequence is the reverse-complement of theselected primer.

TABLE 62 Second strategy for the selection of Streptococcus pyogenes-specific amplification primers from speA sequences. Accession SEQ ID #388                                      427   501                         529NO.: speA X61573TA TGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2466 speA AF029051TA TGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2467 speA X61571TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2468 speA X61570TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2469 speA X61568TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2470 speA X61569TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2471 speA X61572TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2472 speA X61560TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2473 speA U40453TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT 993 speA X61554TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2474 speA X61557TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2475 speA X61559TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2476 speA X61558TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2477 speA X61556TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2478 speA X61555TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2479 speA X61560TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2480 speA X61561TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2481 speA X61566TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2482 speA X61567TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2483 speA X61562TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2484 speA X61563TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2485 speA X61564TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2486 speA X61565TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2487 speA AF055698TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAGACT2488 speA X03929TA CGGAGGGGTA ACAAATCATG AAGGGAATCA TTTAGAAA...AAAAATGGT AACTGCTCAA GAATTAG.CT2489 Selected      GGAGGGGTA ACAAATCATG AAGG  998 sequences for              ACAAATCATG AAGGGAATCA TTTAG  996 species- specific primersSelected                                                   AATGGT AACTGCTCAA GAATTAG 997 sequence for species- specific primer^(a) The sequence numberingrefers to the Streptococcus pyogenes speA gene fragment (SEQ ID NO.993). Dots indicate gaps in the sequences displayed. ^(a)This sequenceis the reverse-complement of the selected primer.

TABLE 63 Strategy for the selection of Streptococcus pyogenes-specificamplification primers from tuf sequences.140                                              186   619                          647SEQ ID NO.: S. anginosusA AGTTGACtTg GTTGACGAtG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATt cAtCCACACA CTAAATT 211 S. bovisA AGTTGACCTT GTTGATGACG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATC cACCCACACA CTAAATT 212 S. dysgalactiaeA AATTGACCTT GTTGAcGAtG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATC AACCCACACA CTAAATT 217 S. pyogenesA AGTTGACCTT GTTGATGACG AAGAGTTGCT TGAATTAGTT GAGATG...CC AAGTTCAATC AACCCACACA CTAAATT1002 S. agalactiaeA AGTTGACCTT GTTGATGAtG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATC AACCCACACA CTAAATT  144^(a) S. oralisA AATTGACtTg GTAGAcGACG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATC AACCCACACA CTAAATT 985 S. pneumoniaeA AGTTGACtTg GTTGAcGACG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATC AACCCACACA CTAAATT  145^(a) S. cristatusA GATCGACtTg GTTGATGACG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATC AACCCACACA CTAAATT 215 S. mitisA GATCGACtTg GTTGATGACG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATC AACCCACACA CTAAATT 982 S. gordoniiA AGTTGACtTg GTTGAcGAtG AAGAaTTGCT TGAGTTgGTT GAaATG...CC AgGTTCAATC AACCCACACA CTAAATT 200 S. sanguinisA AGTTGACtTg GTTGAcGAtG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATC AACCCACACA CTAAATT 227 S. parasanguinisA AGTTGACtTg GTTGATGAtG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATC AACCCACACA CTAAATT 225 S. salivariusA AGTTGACtTg GTTGAcGAtG AAGAaTTGCT TGAATTgGTT GAaATG...CC TgGTTCAATC AACCCACACA CTAAATT  146^(a) S. vestibularisA AGTTGACtTg GTTGAcGAtG AAGAaTTGCT TGAATTgGTT GAaATG.. CC TgGTTCAATC AACCCACACA CTAAATT 231 S. suisA AGTTGACtTg GTTGAcGAtG AAGAaTTGCT TGAgTTgGTT GAaATG...CC AgGTTCtATC AACCCACACA CTAAATT 229 S. mutansA AGTTGAttTg GTTGAcGAtG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATt cACCCACAcA CTAAATT 224 S. rattiA GGTTGACtTg GTTGATGAtG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGTTCAATt cAtCCgCAcA CTAAATT 226 S. macacaeA AGTTGACtTa GTTGATGAtG AAGAaTTGCT TGAATTgGTT GAaATG...CC AgGATCAATt mAtCCACAcA CTAAATT 222 S. cricetusA GGTTGACtTg GTTGAcGAtG AAGAaTTGCT TGAATTgGTT GAaATG...CC TgGTTCAATC cAtCCACACA CTAAATT 214 E. faecalisA AATgGAtaTg GTTGATGACG AAGAaTTatT aGAATTAGTa GAaATG...CC AgcTaCAATC ActCCACACA CaAAATT 607 S. aureusA AGTTGACaTg GTTGAcGAtG AAGAaTTatT aGAATTAGTa GAaATG...CC TgGTTCAATt AcaCCACACA CTgAATT 176 B. cereusA ATgcGACaTg GTaGATGACG AAGAaTTatT aGAATTAGTa GAaATG...AG CgGTTCtgTa AAagCtCACg CTAAATT   7 E. coliA ATgcGACaTg GTTGATGACG AAGAGcTGCT gGAAcTgGTT GAaATG...CC GgGCaCcATC AAgCCgCACA CcAAGTT  78 Selected sequences for species-       TTGACCTT GTTGATGACG AAGAG 999 specific primers                         AAGAGTTGCT TGAATTAGTT GAG1001 Selected sequence for species-                                                          AGTTCAATC AACCCACACA CTAA1000 specific primer^(b) The sequence numbering refers to theStreptococcus pyogenes tuf gene fragment (SEQ ID NO. 1002). Nucleotidesin capitals are identical to the selected sequences or match thosesequences. Mismatches are indicated by lower-case letters. Dots indicategaps in the sequences displayed. ^(a)The SEQ ID NO. refers to previouspatent publication WO98/20157. ^(b)This sequence is thereverse-complement of the selected primer.

TABLE 64 Strategy for the selection stx₁-specific amplification primersand hybridization probe. SEQ ID Accession #230                               263   343                              375   391                            421NO.: stx₁ M19473aTTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCG...TATCG CTTTGCTGAT TTTTCACATG TTACCTTT...GTTACAT TGTCTGGTGA CAGTAGCTAT ACCA2490 stx₁ M16625TTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCG...TATCG CTTTGCTGAT TTTTCACATG TTACCTTT...GTTACAT TGTCTGGTGA CAGTAGCTAT ACCA2491 stx₁ M17358TTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCG...TATCG CTTTGCTGAT TTTTCACATG TTACCTTT...GTTACAT TGTCTGGTGA CAGTAGCTAT ACCA2492 stx₁ Z36900TTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCG...TATCG CTTTGCTGAT TTTTCACATG TTACCTTT...GTTACAT TGTCTaGTGA CAGTAGCTAT ACCA2493 stx₁ L04539TTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCG...TATCG CTTTGCTGAT TTTTCACATG TTACCTTT...GTTACAT TGTCTGGTGA CAGTAGCTAT ACCA2494 stx₁ M19437TTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCG...TATCG CTTTGCTGAT TTTTCACATG TTACCTTT...GTTACAT TGTCTGGTGA CAGTAGCTAT ACCA2495 stx₁ M24352TTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCG...TATCG CTTTGCTGAT TTTTCACATG TTACCTTT...GTTACAT TGTCTGGTGA CAGTAGCTAT ACCA2496 stx₁ X07903TTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCG...TATCG CTTTGCTGAT TTTTCACATG TTACCTTT...GTTACAT TGTCTGGTGA CAGTAGCTAT ACCA2497 stx₁ Z36899TTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCG...TATCG CTTTGCTGAT TTTTCACATG TTACCTTT...GTTACAT TGTCTGGTGA CAGTAGCTAT ACCA2498 stx₁ Z36901TTGATGTC AGAGGGATAG ATCCAGAGGA AGGGCG...TATCG CTTTGCTGAT TTTTCACATG TTACCTTT...GTTACAT TGTCTGGTGA CAGTAGCTAT ACCA1076 stx₂ X61283TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA2499 stx₂ L11079TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA2500 stx₂ M21534TAGgTaTa cGAGGGcTtG ATgtttAtcA gGaGCG...TACaG aTTTtCaGAT TTTgCACATa TatCaTTG...ATTtCca TGaCaacgGA CAGcAGtTAT ACCA2501 stx₂ M36727TAGgTaTa cGAGGGcTtG ATgtttAtcA gGaGCG...TACaG aTTTtCaGAT TTTgCACATa TatCaTTG...ATTtCca TGaCaacgGA CAGcAGtTAT ACCA2502 stx₂ X81415TAGgTaTa cGAGGGcTtG ATgtttAtcA gGaGCG...TACaG aTTTtCaGAT TTTgCACATa TatCaTTG...ATTtCca TGaCaacgGA CAGcAGtTAT ACCA2503 stx₂ X81416TAGgTaTa cGAGGGcTtG ATgtttAtcA gGaGCG...TACaG aTTTtCaGAT TTTgCACATa TatCaTTG...ATTtCca TGaCaacgGA CAGcAGtTAT ACCA2504 stx₂ X81417TAGgTaTa cGAGGGcTtG ATgtttAtcA gGaGCG...TACaG aTTTtCaGAT TTTgCACATa TatCaTTG...ATTtCca TGaCaacgGA CAGcAGtTAT ACCA2505 stx₂ X81418TAGgTaTa cGAGGGcTtG ATgtttAtcA gGaGCG...TACaG aTTTtCaGAT TTTgCACATa TatCaTTG...ATTtCca TGaCaacgGA CAGcAGtTAT ACCA2506 stx₂ E03962TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA2507 stx₂ E03959TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA2508 stx₂ X07865TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA2509 stx₂ Y10775TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA2510 stx₂ Z37725TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA1077 stx₂ Z50754TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA2511 stx₂ X67514TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA2512 stx₂ L11078TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA2513 stx₂ X65949TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACCG tTTTtCaGAT TTTaCACATa TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA2514 stx₂ AF043627TGGATaTa cGAGGGcTtG ATgtctAtcA gGcGCG...TACaG aTTTtCaGAT TTTgCACATa TatCaGTG...GTTtCca TGaCaacgGA CAGcAGtTAT ACCA2515 Selected sequence for    ATGTC AGAGGGATAG ATCCAGAGGA AGG 1081amplification primer Selected sequence for                                          CG CTTTGCTGAT TTTTCACATG TTACC1084 hybridization probe Selected sequence for                                                                                ACAT TGTCTGGTGA CAGTAGCTAT A1080 amplification primer^(a) The sequence numbering refers to theEscherichia coli stx₁ gene fragment (SEQ ID NO. 1076). Nucleotides incapitals are identical to the selected sequences or match thosesequences. Mismatches are indicated by lower-case letters. Dots indicategaps in the sequences displayed. ^(a)This sequence is thereverse-complement of the selected primer.

TABLE 65Strategy for the selection of stx₂-specific amplification primersand hybridization probe. Accession #543                         570   614                            641   684                      708SEQ ID NO.: stx₁ M19473AGCga TgtTaCGgTT TGTtACTGTG ACA...CAAC ACTGgaTGAt ctcAgTGggC gTtcTTA...A AGgtTgAGtA gCGTcCTgCC tGAC2516 stx₁ M16625AGCga TgtTaCGgTT TGTtACTGTG ACA...CAAC ACTGgaTGAt ctcAgTGggC gTtcTTA...A AGgtTgAGtA gCGTcCTgCC tGAC2517 stx₁ M17358AGCga TgtTaCGgTT TGTtACTGTG ACA...CAAC ACTGgaTGAt ctcAgTGggC gTtcTTA...A AGgtTgAGtA gCGTcCTgCC tGAC2518 stx₁ Z36900AGCga TgtTaCGgTT TGTtACTGTG ACA...CAAC ACTGgaTGAt ctcAgTGggC gTtcTTA...A AGgtTgAGtA gTGTeCTgCC tGAT2519 stx₁ L04539AGCga TgtTaCGgTT TGTtACTGTG ACA...CAAC ACTGgaTGAt ctcAgTGggC gTtcTTA...A AGgtTgAGtA gTGTcCTgCC tGAT2520 stx₁ M19437AGCga TgtTaCGgTT TGTtACTGTG ACA...CAAC ACTGgaTGAt ctcAgTGggC gTtcTTA...A AGgtTgAGtA gTGTcCTgCC tGAC2521 stx₁ M24352AGCga TgtTaCGgTT TGTtACTGTG ACA...CAAC ACTGgaTGAt ctcAgTGggC gTtcTTA...A AGgtTgAGtA gTGTcCTgCC tGAC2522 stx₁ X07903AGCga TgtTaCGgTT TGTtACTGTG ACA...CAAC ACTGgaTGAt ctcAgTGggC gTtcTTA...A AGgtTgAGtA gTGTcCTgCC tGAC2523 stx₁ Z36899AGCga TgtTaCGgTT TGTtACTGTG ACA...CAAC ACTGgaTGAt ctcAgTGggC gTtcTTA...A AGgtTgAGtA gTGTeCTgCC tGAC2524 stx₁ Z36901AGCga TgtTaCGgTT TGTtACTGTG ACA...CAAC ACTtgaTGAt ctcAgTGggC gTtcTTA...A AGgtTgAGtA gTGTeCTgCC tGAC1076 stx₂ X61283AGCAG TTCTGCGTTT TGTCACTGTC ACA...AGGC ACTGTCTGA. ..AACTGCTC CTGTGTA...G CGAATCAGCA ATGTGCTTCC GGAG2525 stx₂ L11079AGCAG TTCTGCGTTT TGTCACTGTC ACA...AGGC ACTGTCTGA. ..AACTGCTC CTGTGTA...G CGAATCAGCA ATGTGCTTCC GGAG2526 stx₂ M21534AGCAG TTCTGCGTTT TGTCACTGTC ACA...TGGC ACTGTCTGA. ..AACTGCTC CTGTTTA...G AGAATCAGCA ATGTGCTTCC GGAG2527 stx₂ M36727AGCAG TTCTGCGTTT TGTCACTGTC ACA...TGGC ACTGTCTGA. ..AACTGCTC CTGTTTA...G AGAATCAGCA ATGTGCTTCC GGAG2528 stx₂ U72191AGCAG TTCTGCGTTT TGTCACTGTC ACA...TGGC ACTGTCTGA. ..AACTGCTC CTGTTTA...G AGAATCAGCA ATGTGCTTCC GGAG2529 stx₂ X81415AGCAG TTCTGCGTTT TGTCACTGTC ACA...TGGC ACTGTCTGA. ..AACTGCTC CTGTTTA...G AGAATCAGCA ATGTGCTTCC GGAG2530 stx₂ X81416AGCAG TTCTGCGTTT TGTCACTGTC ACA...TGGC ACTGTCTGA. ..AACTGCTC CTGTTTA...G AGAATCAGCA ATGTGCTTCC GGAG2531 stx₂ X81417AGCAG TTCTGCGTTT TGTCACTGTC ACA...TGGC ACTGTCTGA. ..AACTGCTC CTGTTTA...G AGAATCAGCA ATGTGCTTCC GGAG2532 stx₂ X81418AGCAG TTCTGCGTTT TGTCACTGTC ACA...TGGC ACTGTCTGA. ..AACTGCTC CTGTTTA...G AGAATCAGCA ATGTGCTTCC GGAG2533 stx₂ E03962AGCAG TTCTGCGTTT TGTCACTGTC ACA...AGGC ACTGTCTGA. ..AACTGCTC CTGTGTA...G CGAATCAGCA ATGTGCTTCC GGAG2534 stx₂ E03959AGCAG TTCTGCGTTT TGTCACTGTC ACA...AGGC ACTGTCTGA. ..AACTGCTC CTGTGTA...G CGAATCAGCA ATGTGCTTCC GGAG2535 stx₂ X07865AGCAG TTCTGCGTTT TGTCACTGTC ACA...AGGC ACTGTCTGA. ..AACTGCTC CTGTGTA...G CGAATCAGCA ATGTGCTTCC GGAG2536 stx₂ Y10775AGCAG TTCTGCGTTT TGTCACTGTC ACA...AGGC ACTGTCTGA. ..AACTGCTC CTGTGTA...G CGAATCAGCA ATGTGCTTCC GGAG2537 stx₂ Z37725AGCAG TTCTGCGTTT TGTCACTGTC ACA...AGGC ACTGTCTGA. ..AACTGCTC CTGTGTA...G CGAATCAGCA ATGTGCTTCC GGAG1077 stx₂ Z50754AGCAG TTCTGCGTTT TGTCACTGTC ACA...AGGC ACTGTCTGA. ..AACTGCTC CTGTGTA...G CGAATCAGCA ATGTGCTTCC GGAG2538 stx₂ X67514AGCAG TTCTGCGTTT TGTCACTGTC ACA...AGGC ACTGTCTGA. ..AACTGCTC CTGTGTA...G CGAATCAGCA ATGTGCTTCC GGAG2539 stx₂ L11078AGCAG TTCTGCGTTT TGTCACTGTC ACA...AGGC ACTGTCTGA. ..AACTGCTC CTGTGTA...G AGAATCAGCA ATGTGCTTCC GGAG2540 stx₂ X65949AGCAG TTCTGCGTTT TGTCACTGTC ACA...AGGC ACTGTCTGA. ..AACTGCTC CTGTGTA...G AGAATCAGCA ATGTGCTTCC GGAG2541 stx₂ AF043627AGCAG TTCTGCGTTT TGTCACTGTC ACA...TGGC ACTGTCTGA. ..AACTGCTC CTGTTTA...G AGAATCAGCA ATGTGCTTCC GGAG2542 Selected sequence for    AG TTCTGCGTTT TGTCACTGTC 1078amplification primer Selected sequence for                                     C ACTGTCTGA. ..AACTGCTC CTGT 1085hybridization probe Selected sequence for                                                                           AATCAGCA ATGTGCTTCC G1079 amplification primer^(a) The sequence numbering refers to theEscherichia coli stx₂ gene fragment (SEQ ID NO. 1077). Nucleotides incapitals are identical to the selected sequences or match thosesequences. Mismatches are indicated by lower-case letters. Dots indicategaps in the sequences displayed. ^(a)This sequence is thereverse-complement of the selected primer.

TABLE 66Strategy for the selection of vanA-specific amplification primers from van sequences.Accession #926                        952   1230                     1255SEQ ID NO.: vanA X56895GTCAAT AGCGCGGACG AATTGGACTA C...GT AGAGGTCTAG CCCGTGTGGA TATG 1139 vanAM97297 GTCAAT AGCGCGGACG AATTGGACTA C...GT AGAGGTCTAG CCCGTGTGGA TATG1141 vanA —GTCAAT AGCGCGGACG AATTGGACTA C...GT AGAGGTCTAG CCCGTGTGGA TATG 1051 vanA— GTCAAT AGCGCGGACG AATTGGACTA C...GT AGAGGTCTAG CCCGTGTGGA TATG 1052vanA — GTCAAT AGCGCGGACG AATTGGACTA C...GT AGAGGTCTAG CCCGTGTGGA TATG1053 vanA —GTCAAT AGCGCGGACG AATTGGACTA C...GT AGAGGTCTAG CCCGTGTGGA TATG 1054 vanA— GTCAAT AGCGCGGACG AATTGGACTA C...GT AGAGGTCTAG CCCGTGTGGA TATG 1055vanA — GTCAAT AGCGCGGACG AATTGGACTA C...GT AGAGGTCTAG CCCGTGTGGA TATG1056 vanA —GTCAAT-AGCGCGGACG AATTGGACTA C...GT AGAGGTCTAG CCCGTGTGGA TATG 1057 vanA— GTCAAT AGCGCGGACG AATTGGACTA C...GT AGAGGTCTAG CCCGTGTGGA TATG 1049vanA — GTCAAT AGCGCGGACG AATTGGACTA C...GT AGAGGTCTAG CCCGTGTGGA TATG1050 vanB U94526GTAAAc gGtaCGGAaG AAcTtaACGC T...GC AGAGGgCTtG CCCGTGTtGA TCTT 1117 vanBU94527 GTAAAc AGtaCGGAaG AAcTaaACGC T...GC AGAGGgCTtG CtCGTGTtGA TCTT2543 vanB U94528GTAAAc gGtaCGGAaG AAcTtaACGC T...GC AGAGGgCTtG CCCGTGTtGA TCTT 2544 vanBU94529 GTAAAc gGtaCGGAaG AAcTtaACGC T...GC AGAGGgCTtG CCCGTGTtGA TCTT2545 vanB U94530GTAAAc gGtaCGGAaG AAcTtaACGC T...GC AGAGGgCTtG CCCGTGTtGA TCTT 2546 vanBZ83305 GTAAAc gGtaCGGAaG AAcTtaACGC T...GC AGAGGgCTtG CCCGTGTtGA TCTT2547 vanB U81452GTAAAc gGtaCGGAaG AAcTtaACGC T...GC AGAGGgCTtG CCCGTGTtGA TCTT 2548 vanBU35369 GTAAAc AGtaCGGAaG AAcTaaACGC T...GC AGAGGgCTtG CtCGTGTtGA TCTT2549 vanB U72704GTAAAc gGtaCGGAaG AAcTtaACGC T...GC AGAGGgCTtG CCCGTGTtGA TCTT 2550 vanBL06138 GTAAAc AGtaCGGAaG AAcTaaACGC T...GC AGAGGgCTtG CtCGTGTtGA TCTT2551 vanB L15304GTAAAc gGtaCGGAaG AAcTtaACGC T...GC AGAGGgCTtG CCCGTGTtGA TCTT 2552 vanBU00456 GTAAAc AGtaCGGAaG AAcTaaACGC T...GC AGAGGgCTtG CtCGTGTtGA TCTT2553 vanD AF130997GTAtgc AagGCaGAaG AAcTGcAgGC A...GC AGAGGatTgG CCCGcaTtGA cCTG 2554 vanEAF136925 GTAgAa caaaaaagtG AtTTatAtAA A...GC AaAGGatTAG CgaGaaTcGA cTTT2555 Selected sequence for    AAT AGCGCGGACG AATTGGAC 1090amplification primer Selected sequence for                                     GAGGTCTAG CCCGTGTGGA T 1089amplification primer^(a) The sequence numbering refers to theEnterococcus faecium vanA gene fragment (SEQ ID NO. 1139). Nucleotidesin capitals are identical to the selected sequences or match thosesequences. Mismatches are indicated by lower-case letters. Dots indicategaps in the sequences displayed. ^(a)This sequence is thereverse-complement of the above selected primer.

TABLE 67Strategy for the selection of vanB-specific amplification primers from van sequences.Accession #470                       495   608                       633SEQ ID NO.: vanA X56895A CGCaATtGAA tCgGCAaGAC AATAT...ACG GaATCTTtCG tATtCATCAG GAA 1139 vanAM97297 A CGCaATtGAA tCgGCAaGAC AATAT...ACG GaATCTTtCG tATtCATCAG GAA1141 vanA —A CGCaATtGAA tCgGCAaGAC AATAT...ACG GaATCTTtCG tATtCATCAG GAA 1051 vanA— A CGCaATtGAA tCgGCAaGAC AATAT...ACG GaATCTTtCG tATtCATCAG GAA 1052vanA — A CGCaATtGAA tCgGCAaGAC AATAT...ACG GaATCTTtCG tATtCATCAG GAA1053 vanA —A CGCaATtGAA tCgGCAaGAC AATAT...ACG GaATCTTtCG tATtCATCAG GAA 1054 vanA— A CGCaATtGAA tCgGCAaGAC AATAT...ACG GaATCTTtCG tATtCATCAG GAA 1055vanA — A CGCaATtGAA tCgGCAaGAC AATAT...ACG GaATCTTtCG tATtCATCAG GAA1056 vanA —A CGCaATtGAA tCgGCAaGAC AATAT...ACG GaATCTTtCG tATtCATCAG GAA 1057 vanA— A CGCaATtGAA tCgGCAaGAC AATAT...ACG GaATCTTtCG tATtCATCAG GAA 1049vanA — A CGCaATtGAA tCgGCAaGAC AATAT...ACG GaATCTTtCG tATtCATCAG GAA1050 vanB U94526C TGCGATAGAA GCgGCAGGAC AATAT...ACG GTATCTTCCG CATCCATCAG GAA 1117 vanBU94527 C TGCGATAGAA GCAGCAGGAC AATAT...ACG GTATCTTCCG CATCCATCAG GAA2556 vanB U94528C TGCGATAGAA GCgGCAGGAC AATAT...ACG GTATCTTCCG CATCCATCAG GAA 2557 vanBU94529 C TGCGATAGAA GCgGCAGGAC AATAT...ACG GTATCTTCCG CATCCATCAG GAA2558 vanB U94530C TGCGATAGAA GCgGCAGGAC AATAT...ACG GTATCTTCCG CATCCATCAG GAA 2559 vanBZ83305 C TGCGATAGAA GCgGCAGGAC AATAT...ACG GTATCTTCCG CATCCATCAG GAA2560 vanB U81452C TGCGATAGAA GCgGCAGGAC AATAT...ACG GTATCTTCCG CATCCATCAG GAA 2561 vanBU35369 C TGCGATAGAA GCAGCAGGAC AATAT...ACG GTATCTTCCG CATCCATCAG GAA2562 vanB U72704C TGCGATAGAA GCgGCAGGAC AATAT...ATG GTATCTTCCG CATCCATCAG GAA 2563 vanBL06138 C TGCGATAGAA GCAGCAGGAC AATAT...ACG GTATCTTCCG CATCCATCAG GAA2564 vanB L15304C TGCGATAGAA GCgGCAGGAC AATAT...ACG GTATCTTCCG CATCCATCAG GAA 2565 vanBU00456 C TGCGATAGAA GCAGCAGGAC AATAT...ACG GTATCTTCCG CATCCATCAG GAA2566 vanD AF130997C AGCaATcGAA GaAGCAaGAa AATAT...ACG GctTtTTtaa gATtCATCAG GAA 2567 vanEAF136925 A AGCaATAGAc GaAGCttcAa AATAT...ATG GctTtTTCga CtatgAagAG AAA2568 Selected sequence for     CGATAGAA GCAGCAGGAC AA 1095amplification primer Selected sequence for                                    GTATCTTCCG CATCCATCAG 1096amplification primer^(a) The sequence numbering refers to theEnterococcus faecium vanB gene fragment (SEQ ID NO. 1117). Nucleotidesin capitals are identical to the selected sequences or match thosesequences. Mismatches are indicated by lower-case letters. Dots indicategaps in the sequences displayed. ^(a)This sequence is thereverse-complement of the above vanB sequence.

TABLE 68Strategy for the selection of vanC-specific amplification primers from vanC sequences.Accession #929                          957   1064                         1092SEQ ID NO.: vanC1 —GT CGACGGTTTT TTTGATTTTG AAGAGAA...ACGGGTC TGGCTCGAAT CGATTTTTTC GT 1058vanC1 —GT CGACGGTTTT TTTGATTTTG AAGAGAA...ACGGGTC TGGCTCGAAT CGATTTTTTC GT 1059vanC1 M75132GT CGACGGTTTT TTTGATTTTG AAGAGAA...ACGGGTC TGGCTCGAAT CGATTTTTTC GT 1138vanC2 —GT AGACGGCTTT TTCGATTTTG AAGAAAA...AAAGGTC TTGCTCGCAT CGACTTTTTT GT 1060vanC2 —GT AGACGGCTTT TTCGATTTTG AAGAAAA...AAAGGTC TTGCTCGCAT CGACTTTTTT GT 1061vanC2 —GT AGACGGCTTT TTCGATTTTG AAGAAAA...AAAGGTC TTGCTCGCAT CGACTTTTTT GT 1062vanC2 —GT AGACGGCTTT TTCGATTTTG AAGAAAA...AAAGGTC TTGCTCGCAT CGACTTTTTT GT 1063vanC2 L29638GT AGACGGCTTT TTCGATTTTG AAGAAAA...AAAGGTC TTGCTCGCAT CGACTTTTTT GT 2569vanC2 L29638GT AGACGGCTTT TTCGATTTTG AAGAAAA...AAAGGTC TTGCTCGCAT CGACTTTTTT GT 2570vanC3 —GT AGACGGCTTT TTCGATTTTG AAGAAAA...AAAGGTC TTGCTCGCAT CGACTTTTTT GT 1064vanC3 —GT AGACGGCTTT TTCGATTTTG AAGAAAA...AAAGGTC TTGCTCGCAT CGACTTTTTT GT 1065vanC3 —GT AGACGGCTTT TTCGATTTTG AAGAAAA...AAAGGaC TTGCTCGCAT CGACTTTTTT GT 1066vanC3 L29639GT AGACGGCTTT TTCGATTTTG AAGAAAA...AAAGGTC TTGCTCGCAT CGACTTTTTT GT 2571Selected sequence      GACGGYTTT TTYGATTTTG AAGA 1101for resistance primer Selected sequence                                      GGTC TKGCTCGMAT CGAYTTTTT 1102for resistance primer^(a) The sequence numbering refers to the vanC1gene fragment (SEQ ID NO. 1138). Nucleotides in capitals are identicalto the selected sequences or match those sequences. Mismatches areindicated by lower-case letters. Dots indicate gaps in the sequencedisplayed. “R” “Y” “M” “K” “W” and “S” designate nucleotide positionswhich are degenerated. “R” stands for A or G; “Y” stands for C or T; “M”stands for A or C; “K” stands for G or T; “W” stands for A or T; “S”stands for C or G. “I” stands for inosine which is a nucleotide analogthat can bind to any of the four nucleotides A, C, G or T. ^(a)Thissequence is the reverse-complement of the selected sequence.

TABLE 69 Strategy for the selection of Streptococcus pneumoniae-specificamplification primers and hybridization probes from pbp1a sequences.Accession #453                                                     505   678                          706SEQ ID NO.: pbp1a M90528A TTGACTAcCC AAGCATaCAc TATGCtAAtG CtATTTCAAG TAATACAACC GA...TATATG ATGACaGAtA TGATGAAAAC CGT...2572 pbp1a X67873A TCGACTAcCC AAGtATtCAc TActCAAAtG CCATTTCAAG TAAcACAACC GA...TATATG ATGACCGAAA TGATGAAAAC AGT...2573 pbp1a AB006868A TCGACTAcCC AAGtATtCAc TActCAAAtG CCATTTCAAG TAAcACAACC GA...TATATG ATGACCGACA TGATGAAAAC AGT...2574 pbp1a AF046234A TCGACTAcCC AAGtATtCAc TActCAAAtG CCATTTCAAG TAAcACAACC GA...TATATG ATGACCGAAA TGATGAAAAC TGT...2575 pbp1aA TCGACTAcCC AAGtATtCAc TActCAAAtG CCATTTCAAG TAAcACAACC GA...TATATG ATGACCGACA TGATGAAAAC TGT...1014 pbp1aA TCGACTAcCC AAGtATtCAc TActCAAAtG CCATTTCAAG TAAcACAACC GA...TACATG ATGACCGAAA TGATGAAAAC TGT...1017 pbp1a AB006873A TCGACTAcCC AAGtcTtCAc TActCAAAtG CCATTTCAAG TAAcACAACC GA...TATATG ATGACCGACA TGATGAAAAC AGT...2576 pbp1a AF139883A TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA GA...TATATG ATGACCGACA TGATGAAAAC AGT...1169 pbp1aA TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA GA...TATATG ATGACCGACA TGATGAAAAC AGT...1004 pbp1aA TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA GA...TATATG ATGACCGACA TGATGAAAAC AGT...1007 pbp1aA TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA GA...TATATG ATGACCGACA TGATGAAAAC AGT...1008 pbp1aA TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA GA...TATATG ATGACCGACA TGATGAAAAC AGT...1009 pbp1aA TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA GA...TATATG ATGACCGACA TGATGAAAAC AGT...1011 pbp1a AF159448A TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA GA...TATATG ATGACCGACA TGATGAAAAC AGT...2577 pbp1aA TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA GA...TACATG ATGACCGAAA TGATGAAAAC TGT...1005 pbp1aA TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA GA...TACATG ATGACCGAAA TGATGAAAAC TGT...1015 pbp1aA TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA GA...TACATG ATGACCGAAA TGATGAAAAC TGT...1006 pbp1aA TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA GA...TACATG ATGACCGAAA TGATGAAAAC TGT...1012 pbp1a X67867A TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAATACAACA GA...TACATG ATGACCGAAA TGATGAAAAC TGT...2578 pbp1aA TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAAcACAACT GA...TATATG ATGACtGAAA TGATGAAAAC TGT...1010 pbp1a Z49094A TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAAcACAACT GA...TATATG ATGACtGAAA TGATGAAAAC TGT...2579 pbp1aA TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAAcACAACT GA...TATATG ATGACtGAAA TGATGAAAAC TGT...1013 pbp1aA TCGACTATCC AAGCATGCAT TATGCAAACG CCATTTCAAG TAAcACAACT GA...TATATG ATGACtGAAA TGATGAAAAC TGT...1016 pbp1a X67870A TCGACTATCC AAGCATGCAT TAcGCAAACG CCATTTCAAG TAAcACAACT GA...TATATG ATGACCGAAA TGATGAAAAC TGT...2580 pbp1aA TTGACTATCC AAGtATtCAc TActCAAAtG CtATTTCAAG TAATACAACT GA...TATATG ATGACtGAAA TGATGAAAAC TGT...1018 pbp1a AJ002290A TTGAtTAcCC AActATGgtc TATGCtAACG CtATTTCAAG TAATACAACT GA...TACATG ATGACtGAAA TGATGAAAAC AGT...2581 pbp1a X67871A TCGACTAcCC AAGtcTtCAc TActCAAAtG CCATTTCAAG TAAcACAACC GA...TACATG ATGACaGAAA TGATGAAAAC AGT...2582 Selected sequences for     GACTATCC AAGCATGCAT TATG 1130amplification primers                                                                 ATG ATGACHGAMA TGATGAAAAC1129 Selected sequence for                            CAAACG CCATTTCAAG TAATACAAC 1197hybridization probeThe sequence numbering refers to the Streptococcus pneumoniae pbp1a gene fragment (SEQ ID NO. 1004). Nucleotides in capitals are identical to the selected sequences or match those sequences. Mismatches are indicated by lower-case letters. Dotes indicate gaps in the sequences displayed. “R” “Y” “M” “K” “W”and “S” designate nucleotide positions which are degenerated. “R”stands for A or G; “Y” stands for C or T; “M” stands for A or C; “K”stands for G or T; “W” stands for A or T; “H” stands for A, C or T;  “S”stands for C or G. “I”stands for inosine which is a nucleotide analog that can bind to any of the four nucleotides A, C, G or T.Accession #   756                        783   813                         840SEQ ID NO.: pbp1a M90528...GCTGGTAA aACtGGTACg TCaAACTATA...A ATACgGGTTA TGTAGCTCCG GAcGAAA 2583pbp1a X67873...GCTGGTAA aACAGGaACc TCTAACTATA...A CCtCTcaaTt TGTAGCaCCt GATGAAC 2584pbp1a AB006868...GCTGGTAA aACAGGaACc TCTAACTATA...A CCtCTcaaTt TGTAGCaCCt GAcGAAC 2585pbp1a AF046234...GCAGGTAA aACAGGTACT TCTAACTATA...A ACACTGGTTA CGTAGCTCCA GATGAAA 2586pbp1a...GCAGGTAA aACAGGTACT TCTAACTATA...A ACACTGGTTA CGTAGCTCCA GATGAAA 1014pbp1a...GCTGGTAA GACAGGTACT TCTAACTACA...A ACACTGGCTA TGTAGCTCCA GATGAAA 1017pbp1a AB006873...GCAGGTAA GACAGGTACT TCTAACTATA...A ACACTGGCTA CGTAGCTCCA GATGAAA 2587pbp1a AF139883...GCTGGTAA aACAGGaACc TCTAACTATA...A ACACTGGCTA TGTAGCTCCA GATGAAA 1169pbp1a...GCTGGTAA aACAGGaACc TCTAACTATA...A ACACTGGCTA TGTAGCTCCA GATGAAA 1004pbp1a...GCTGGTAA aACAGGaACc TCTAACTATA...A ACACTGGCTA TGTAGCTCCA GATGAAA 1007pbp1a...GCTGGTAA aACAGGaACc TCTAACTATA...A ACACTGGCTA TGTAGCTCCA GATGAAA 1008pbp1a...GCTGGTAA aACAGGaACc TCTAACTATA...A ACACTGGCTA TGTAGCTCCA GATGAAA 1009pbp1a...GCTGGTAA aACAGGaACc TCTAACTATA...A ACACTGGCTA TGTAGCTCCA GATGAAA 1011pbp1a AF159448...GCTGGTAA aACAGGaACc TCTAACTATA...A ACACTGGCTA TGTAGCTCCA GATGAAA 2588pbp1a...GCTGGTAA GACAGGTACT TCTAACTACA...A ACACTGGCTA TGTAGCTCCA GATGAAA 1005pbp1a...GCTGGTAA GACAGGTACT TCTAACTACA...A ACACTGGCTA TGTAGCTCCA GATGAAA 1015pbp1a...GCTGGTAA GACAGGTACT TCTAACTACA...A ACACTGGCTA TGTAGCTCCA GATGAAA 1006pbp1a...GCTGGTAA GACAGGTACT TCTAACTACA...A ACACTGGCTA TGTAGCTCCA GATGAAA 1012pbp1a X67867...GCTGGTAA GACAGGTACT TCTAACTACA...A ACACTGGCTA TGTAGCTCCA GATGAAA 2589pbp1a...GCAGGTAA GACAGGTACT TCTAACTATA...A ACACTGGTTA CGTAGCTCCA GATGAAA 1010pbp1a Z49094...GCAGGTAA GACAGGTACT TCTAACTATA...A ACACTGGTTA CGTAGCTCCA GATGAAA 2590pbp1a...GCAGGTAA GACAGGTACT TCTAACTATA...A ACACTGGCTA CGTAGCTCCA GATGAAA 1013pbp1a...GCAGGTAA GACAGGTACT TCTAACTATA...A ACACTGGCTA CGTAGCTCCA GATGAAA 1016pbp1a X67870...GCAGGTAA GACAGGTACT TCTAACTATA...A ACACTGGCTA CGTAGCTCCA GATGAAA 2591pbp1a...GCAGGTAA GACAGGTACT TCTAACTATA...A ACACTGGCTA CGTAGCTCCA GATGAAA 1018pbp1a AJ002290...GCAGGTAA GACgGGTACa TCTAACTACA...A ACACTGGCTA C~~~~~~~~~ ~~~~~~~ 2592pbp1a X67871...GCTGGTAA aACAGGTACc TCTAACTATA...A ACACTGGTTA CGTAGCTCCA GATGAAA 2593Selected sequence for       GGTAA GACAGGTACT TCTAACT 1193hybridization probe Selected sequence for                                        ACTGGYTA YGTAGCTCCA GATG 1131amplification primer^(a)The sequence numbering refers to the Streptococcus pneumoniae pbp1a gene fragment (SEQ ID NO. 1004). Nucleotides in capitals are identical to the selected sequences or match those sequences. Mismatches are indicated by lower-case letters. Dots indicate gaps in the sequences displayed. “~”indicates incomplete sequence data. “R” “Y” “W” and “S”designate nucleotide positions which are degenerated. “R”stands for A or G; “Y” stands for C or T;  “W” stands for A or T; “S”stands for C or G. “I”stands for inosine which is a nucleotide analog that can bind to any of the four nucleotides A, C, G or T.^(a)This sequence is the reverse-complement of the selected primer.

TABLE 70 Specific and ubiquitous primers fornucleic acid amplification (toxin sequences). Originating DNA fragmentSEQ ID Nucleotide SEQ ID NO. Nucleotide sequence NO. positionToxin gene: cdtA 2123 5′-TCT ACC ACT GAA GCA TTA C 2129^(a) 442-4602124^(b) 5′-TAG GTA CTG TAG GTT TAT TG 2129^(a) 580-599 Toxin gene: cdtB2126 5′-ATA TCA GAG ACT GAT GAG 2130^(a) 2665-2682 2127^(b)5′-TAG CAT ATT CAG AGA ATA TTG T 2130^(a) 2746-2767 Toxin gene: stx₁1081 5′-ATG TCA GAG GGA TAG ATC CA 1076^(a) 233-252 1080^(b)5′-TAT AGC TAC TGT CAC CAG ACA ATG T 1076^(a) 394-418 Toxin gene: stx₂1078 5′-AGT TCT GCG TTT TGT CAC TGT C 1077^(a) 546-567 1079^(b)5′-CGG AAG CAC ATT GCT GAT T 1077^(a) 687-705 Toxin genes: stx₁ and stx₂1082 5′-TTG ARC RAA ATA ATT TAT ATG TG 1076^(a) 278-300 1083^(b)5′-TGA TGA TGR CAA TTC AGT AT 1076^(a) 781-800 ^(a)Sequences fromdatabases. ^(b)These sequences are from the complementary DNA strand ofthe sequence of the originating fragment given in the Sequence Listing.

TABLE 71 Molecular beacon internal hybridization probes for specificdetection of toxin sequences. Originating DNA fragment NucleotideSEQ ID NO. Nucleotide sequence^(a) SEQ ID NO. position Toxin gene:                 cdtA 2125^(b) 5′-CAC GCG GAT TTT GAA TCT CTT CCT CTA2129^(c) 462-488    GTA GCG CGT G Toxin gene:                  cdtB 21285′-CAA CGC TGG AGA ATC TAT ATT TGT AGA 2130^(c) 2714-2740   AAC TGC GTT G Toxin gene:                  stx₁ 10845′-CCA CGC CGC TTT GCT GAT TTT TCA CAT 1076^(c) 337-363   GTT ACC GCG TGG 2012^(d) 5′-CCG CGG ATT ATT AAA CCG CCC TTC CGC1076^(c) 248-264    GG-MR-HEG-ATG TCA GAG GGA TAG ATC CA Toxin gene:                 stx₂ 1085 5′-CCA CGC CAC TGT CTG AAA CTG CTC CTG1077^(c) 617-638    TG CGT GG ^(a)Underlined nucleotides indicate themolecular beacon's stem. ^(b)These sequences are from the complementaryDNA strand of the sequence of the originating fragment given in theSequence Listing. ^(c)Sequences from databases. ^(d)Scorpion primer.

TABLE 72Specific and ubiquitous primers for nucleic acid amplification (van sequences).Originating DNA fragment Nucleotide SEQ ID NO. Nucleotide sequenceSEQ ID NO. position Resistance gene:            vanA 10865′-CTA CTC CCG CCT TTT GGG TT 1049-1057^(a)  513-532^(b) 1087^(c)5′-CTC ACA GCC CGA AAC AGC CT 1049-1057^(a)  699-718^(b) 10865′-CTA CTC CCG CCT TTT GGG TT 1049-1057^(a)  513-532^(b) 1088^(c)5′-TGC CGT TTC CTG TAT CCG TC 1049-1057^(a)  885-904^(b) 10865′-CTA CTC CCG CCT TTT GGG TT 1049-1057^(a)  513-532^(b) 1089^(c)5′-ATC CAC ACG GGC TAG ACC TC 1049-1057^(a)   933-952^(b) 10905′-AAT AGC GCG GAC GAA TTG GAC 1049-1057^(a)  629-649^(b) 1091^(c)5′-AAC GCG GCA CTG TTT CCC AA 1049-1057^(a)  734-753^(b) 10905′-AAT AGC GCG GAC GAA TTG GAC 1049-1057^(a)  629-649^(b) 1089^(c)5′-ATC CAC ACG GGC TAG ACC TC 1049-1057^(a)  933-952^(b) 10925′-TCG GCA AGA CAA TAT GAC AGC 1049-1057^(a)  662-682^(b) 1088^(c)5′-TGC CGT TTC CTG TAT CCG TC 1049-1057^(a)  885-904^(b)Resistance gene:            vanB 1095 5′-CGA TAG AAG CAG CAG GAC AA1117^(d)  473-492 1096^(c) 5′-CTG ATG GAT GCG GAA GAT AC 1117^(d) 611-630 Resistance genes:           vanA, vanB 11125′-GGC TGY GAT ATT CAA AGC TC 1049-1057, 1117^(a)  437-456^(b) 1113^(c)5′-ACC GAC CTC ACA GCC CGA AA 1049-1057, 1117^(a)  705-724^(b) 11125′-GGC TGY GAT ATT CAA AGC TC 1049-1057, 1117^(a)  437-456^(b) 1114^(c)5′-TCW GAG CCT TTT TCC GGC TCG 1049-1057, 1117^(a)   817-837^(b) 11155′-TTT CGG GCT GTG AGG TCG GBT GHG CG 1049-1057, 1117^(a)  705-730^(b)1114^(c) 5′-TCW GAG CCT TTT TCC GGC TCG 1049-1057, 1117^(a)  817-837^(b)1116 5′-TTT CGG GCT GTG AGG TCG GBT GHG CGG  1049-1057, 1117^(a) 705-731^(b) 1114^(c) 5′-TCW GAG CCT TTT TCC GGC TCG 1049-1057, 1117^(a) 817-837^(b) 1112 5′-GGC TGY GAT ATT CAA AGC TC 1049-1057, 1117^(a) 437-456^(b) 1118^(c) 5′-TTT TCW GAG CCT TTT TCC GGC TCG1049-1057, 1117^(a)  817-840^(b)^(a)These sequences were aligned to derive the corresponding primer. ^(b)The nucleotide positions refer to the vanA sequence fragment (SEQ ID NO. 1051). ^(c)These sequences are from the complementary DNA strand of the sequence of the originating fragment given in the Sequence Listing.^(d)Sequences from databases.  Resistance genes:           vanA, vanB1115 5′-TTT CGG GCT GTG AGG TCG GBT GHG CG 1049-1057, 1117^(a) 705-730^(b) 1118^(c) 5′-TTT TCW GAG CCT TTT TCC GGC TCG1049-1057, 1117^(a)  817-840^(b) 11165′-TTT CGG GCT GTG AGG TCG GBT GHG CGG  1049-1057, 1117^(a)  705-731^(b)1118^(c) 5′-TTT TCW GAG CCT TTT TCC GGC TCG 1049-1057, 1117^(a) 817-840^(b) 1119 5′-TTT CGG GCT GTG AGG TCG GBT GHG C1049-1057, 1117^(a)  705-729^(b) 1118^(c)5′-TTT TCW GAG CCT TTT TCC GGC TCG 1049-1057, 1117^(a)  817-840^(b) 11205′-TTT CGG GCT GTG AGG TCG GBT GHG 1049-1057, 1117^(a)  705-728^(b)1118^(c) 5′-TTT TCW GAG CCT TTT TCC GGC TCG 1049-1057, 1117^(a) 817-840^(b) 1121 5′-TGT TTG WAT TGT CYG GYA TCC C 1049-1057, 1117^(a) 408-429^(b) 1111^(c) 5′-CTT TTT CCG GCT CGW YTT CCT GAT G1049-1057, 1117^(a)  806-830^(b) 1112 5′-GGC TGY GAT ATT CAA AGC TC1049-1057, 1117^(a)  437-456^(b) 1111^(c)5′-CTT TTT CCG GCT CGW YTT CCT GAT G 1049-1057, 1117^(a)  806-830^(b)1123 5′-TTT CGG GCT GTG AGG TCG GBT G 1049-1057, 1117^(a)  705-726^(b)1111^(c) 5′-CTT TTT CCG GCT CGW YTT CCT GAT G 1049-1057, 1117^(a) 806-830^(b) 1112 5′-GGC TGY GAT ATT CAA AGC TC 1049-1057, 1117^(a) 437-456^(b) 1124^(c) 5′-GAT TTG RTC CAC YTC GCC RAC A1049-1057, 1117^(a)  757-778^(b) Resistance gene:            vanC1 11035′-ATC CCG CTA TGA AAA CGA TC 1058-1059^(a)  519-538^(d) 1104^(c)5′-GGA TCA ACA CAG TAG AAC CG 1058-1059^(a)  678-697^(d)Resistance genes:           vanC1, vanC2, vanC3 10975′-TCY TCA AAA GGG ATC ACW AAA GTM AC 1058-1066^(a)  607-632^(d)1098^(c) 5′-TCT TCA AAA TCG AAA AAG CCG TC 1058-1066^(a)  787-809^(d)1099 5′-TCA AAA GGG ATC ACW AAA GTM AC 1058-1066^(a)  610-632^(d)1100^(c) 5′-GTA AAK CCC GGC ATR GTR TTG ATT TC 1058-1066^(a) 976-1001^(d) 1101 5′-GAC GGY TTT TTY GAT TTT GAA GA 1058-1066^(a) 787-809^(d) 1102^(c) 5′-AAA AAR TCG ATK CGA GCM AGA CC 1058-1066^(a) 922-944^(d) Resistance genes:           vanC2, vanC3 11055′-CTC CTA CGA TTC TCT TGA YAA ATC A 1060-1066, 1140^(a)  487-511^(e)1106^(c) 5′-CAA CCG ATC TCA ACA CCG GCA AT 1060-1066, 1140^(a) 690-712^(e)^(a)These sequences were aligned to derive the corresponding primer. ^(b)The nucleotide positions refer to the vanA sequence fragment (SEQ ID NO. 1051). ^(c)These sequences are from the complementary DNA strand of the sequence of the originating fragment given in the Sequence Listing.^(d)The nucleotide positions refer to the vanC1 sequence fragment (SEQ ID NO. 1058). ^(e)The nucleotide positions refer to the vanC2 sequence fragment (SEQ ID NO. 1140). Resistance gene:            vanD 1591 5′-ATG AGG TAA TAG AAC GGA TT 1594 797-837 1592^(b) 5′-CAG TAT TTC AGT AAG CGT AAA 1594  979-999Resistance gene:            vanE 15955′-AAA TAA TGC TCC ATC AAT TTG CTG A 1599^(a)   74-98 1596^(b)5′-ATA GTC GAA AAA GCC ATC CAC AAG 1599^(a)  394-417 15975′-GAT GAA TTT GCG AAA ATA CAT GGA 1599^(a)  163-186 1598^(b)5′-CAG CCA ATT TCT ACC CCT TTC AC 1599^(a)  319-341                 Sequencing primers (vanAB) 11125′-GGC TGY GAT ATT CAA AGC TC 1139^(a)  737-756 1111^(b)5′-CTT TTT CCG GCT CGW YTT CCT GAT G 1139^(a) 1106-1130                 Sequencing primers (vanA, vanX, vanY) 11505′-TGA TAA TCA CAC CGC ATA CG 1141^(a)  860-879 1151^(b)5′-TGC TGT CAT ATT GTC TTG CC 1141^(a) 1549-1568 11525′-ATA AAG ATG ATA GGC CGG TG 1141^(a) 1422-1441 1153^(b)5′-CTC GTA TGT CCC TAC AAT GC 1141^(a) 2114-2133 11545′-GTT TGA AGC ATA TAG CCT CG 1141^(a) 2520-2539 1155^(b)5′-CAG TGC TTC ATT AAC GTA GTC 1141^(a) 3089-3109                 Sequencing primers (vanC1) 11105′-ACG AGA AAG ACA ACA GGA AGA CC 1138^(a)  122-144 1109^(b)5′-ACA TCG TGA TCG CTA AAA GGA GC 1138^(a) 1315-1337                  Sequencing primers (vanC2, vanC3) 11085′-GTA AGA ATC GGA AAA GCG GAA GG 1140^(a)    1-23 1107^(b)5′-CTC ATT TGA CTT CCT CCT TTG CT 1140^(a) 1064-1086 ^(a)Sequences fromdatabases. ^(b)These sequences are from the complementary DNA strand ofthe sequence of the originating fragment given in the Sequence Listing.

TABLE 73Internal hybridization probes for specific detection of van sequences.Originating DNA fragment SEQ ID NO. Nucleotide sequence SEQ ID NO.Nucleotide position Resistance gene:                 vanA 11705′-ACG AAT TGG ACT ACG CAA TT 1049-1057^(a) 639-658^(b) 22925′-GAA TCG GCA AGA CAA TAT G 2293^(c) 583-601Resistance gene:                 vanB 1171 5′-ACG AGG ATG ATT TGA TTG TC1117^(c) 560-579 2294 5′-AAA CGA GGA TGA TTT GAT TG 2296^(a) 660-6792295 5′-TTG AGC AAG CGA TTT CGG 2296^(a) 614-631Resistance gene:                 vanD 2297 5′-TTC AGG AGG GGG ATC GC1594^(c) 458-474 ^(a)These sequences were aligned to derive thecorresponding primer. ^(b)The nucleotide positions refer to the vanAsequence fragment (SEQ ID NO. 1051). ^(c)Sequences from databases.

TABLE 74Specific and ubiquitous primers for nucleic acid amplification (pbp sequences).Originating DNA fragment Nucleotide SEQ ID NO. Nucleotide sequenceSEQ ID NO. position Resistance gene:        pbp1a 11295′-ATG ATG ACH GAM ATG ATG AAA AC 1004-1018^(a) 681-703^(b) 1131^(c)5′-CAT CTG GAG CTA CRT ARC CAG T 1004-1018^(a) 816-837^(b) 11305′-GAC TAT CCA AGC ATG CAT TAT G 1004-1018^(a) 456-477^(b) 11315′-CAT CTG GAG CTA CRT ARC CAG T 1004-1018^(a) 816-837^(b) 20155′-CCA AGA AGC TCA AAA ACA TCT G 2047^(d) 909-930 2016^(c)5′-TAD CCT GTC CAW ACA GCC AT 2047^(d) 1777-1796            Sequencing primers (pbp1a) 11255′-ACT CAC AAC TGG GAT GGA TG 1169^(d) 873-892 1126^(c)5′-TTA TGG TTG TGC TGG TTG AGG 1169^(d) 2140-2160 11255′-ACT CAC AAC TGG GAT GGA TG 1169^(d) 873-892 1128^(c)5′-GAC GAC YTT ATK GAT ATA CA 1169^(d) 1499-1518 11275′-KCA AAY GCC ATT TCA AGT AA 1169^(d) 1384-1403 1126^(c)5′-TTA TGG TTG TGC TGG TTG AGG 1169^(d) 2140-2160            Sequencing primers (pbp2b) 11425′-GAT CCT CTA AAT GAT TCT CAG GTG G 1172^(d)  1-25 1143^(c)5′-CAA TTA GCT TAG CAA TAG GTG TTG G 1172^(d) 1481-1505 11425′-GAT CCT CTA AAT GAT TCT CAG GTG G 1172^(d)  1-25 1145^(c)5′-AAC ATA TTK GGT TGA TAG GT 1172^(d) 793-812 11445′-TGT YTT CCA AGG TTC AGC TC 1172^(d) 657-676 1143^(c)5′-CAA TTA GCT TAG CAA TAG GTG TTG G 1172^(d) 1481-1505            Sequencing primers (pbp2x) 11465′-GGG ATT ACC TAT GCC AAT ATG AT 1173^(d) 219-241 1147^(c)5′-AGC TGT GTT AGC VCG AAC ATC TTG 1173^(d) 1938-1961 11465′-GGG ATT ACC TAT GCC AAT ATG AT 1173^(d) 219-241 1149^(c)5′-TCC YAC WAT TTC TTT TTG WG 1173^(d) 1231-1250 11485′-GAC TTT GTT TGG CGT GAT AT 1173^(d) 711-730 1147^(c)5′-AGC TGT GTT AGC VCG AAC ATC TTG 1173^(d) 1938-1961 ^(a)Thesesequences were aligned to derive the corresponding primer. ^(b)Thenucleotide positions refer to the pbp1a sequence fragment (SEQ ID NO.1004). ^(c)These sequences are from the complementary DNA strand of thesequence of the originating fragment given in the Sequence Listing.^(d)Sequences from databases.

TABLE 75Internal hybridization probes for specific detection of pbp sequences.Originating DNA fragment SEQ ID Nucleotide SEQ ID NO.Nucleotide sequence NO. position Resistance gene:       pbp1a 11325′-AGT GAA AAR ATG GCT GCT GC 1004-1018^(a) 531-550^(b) 11335′-CAT CAA GAA CAC TGG CTA YGT AG 1004-1018^(a) 806-828^(b) 11345′-CTA GAT AGA GCT AAA ACC TTC CT 1004-1018^(a) 417-439^(b) 11355′-CAT TAT GCA AAC GCC ATT TCA AG 1004-1018^(a) 471-493^(b) 11925′-GGT AAA ACA GGA ACC TCT AAC T 1004-1018^(a) 759-780^(b) 11935′-GGT AAG ACA GGT ACT TCT AAC T 1004-1018^(a) 759-780^(b) 11945′-CAT TTC AAG TAA TAC AAC AGA ATC 1004-1018^(a) 485-508^(b) 11955′-CAT TTC AAG TAA CAC AAC TGA ATC 1004-1018^(a) 485-508^(b) 11965′-GCC ATT TCA AGT AAT ACA ACA GAA 1004-1018^(a) 483-506^(b) 11975′-CAA ACG CCA TTT CAA GTA ATA CAA C 1004-1018^(a) 478-502^(b) 10945′-GGT AAA ACA GGT ACT TCT AAC TA 1004-1018^(a) 759-781^(b) 12145′-GGT AAA ACA GGT ACC TCT AAC TA 1004-1018^(a) 759-781^(b) 12165′-GGT AAG ACT GGT ACA TCA AAC TA 1004-1018^(a) 759-781^(b) 12175′-CAA ATG CCA TTT CAA GTA ACA CAA C 1004-1018^(a) 478-502^(b) 12185′-CAA ACG CCA TTT CAA GTA ACA CAA C 1004-1018^(a) 478-502^(b) 12195′-CAA ATG CTA TTT CAA GTA ATA CAA C 1004-1018^(a) 478-502^(b) 12205′-CAA ACG CCA TTT CAA GTA ATA CGA C 1004-1018^(a) 478-502^(b) 20175′-ACT TTG AAT AAG GTC GGT CTA G  2047^(c) 1306-1327 20185′-ACA CTA AAC AAG GTT GGT TTA G 2063 354-375 20195′-ACA CTA AAC AAG GTC GGT CTA G 2064 346-367 20205′-GTA GCT CCA GAT GAA ATG TTT G  2140^(c) 1732-1753 20215′-GTA GCT CCA GAC GAA ATG TTT G 2057 831-852 20225′-GTA GCT CCA GAT GAA ACG TTT G  2053^(c) 805-826 20235′-GTA ACT CCA GAT GAA ATG TTT G 2056 819-840 20245′-AGT GAA AAG ATG GCT GCT GC  2048^(c) 1438-1457 20255′-AGT GAG AAA ATG GCT GCT GC  2047^(c) 1438-1457 20265′-TCC AAG CAT GCA TTA TGC AAA CG  2047^(c) 1368-1390 20275′-TCG GTC TAG ATA GAG CTA AAA CG  2047^(c) 1319-1341 20285′-TAT GCT CTT CAA CAA TCA CG  2047^(c) 1267-1286 20295′-AGC CGT TGA GAC TTT GAA TAA G  2047^(c) 1296-1317 20305′-CTT AAT GGT CTT GGT ATC G  2047^(c) 1345-1366 20315′-CGT GAC TGG GGT TCT GCT ATG A  2049^(c) 1096-1117 20325′-CGT GAC TGG GGA TCA TCA ATG A  2047^(c) 1096-1117 20335′-CGT GAC TGG GGT TCT GCC ATG A 2057   195-216 20345′-ATC AAG AAC ACT GGC TAT GTA G  2050^(c) 787-808 20355′-ATC AAG AAC ACT GGC TAC GTA G  2051^(c) 787-808 20365′-ATC AAG AAC ACT GGT TAC GTA G 2047 1714-1735 20375′-ATC AAA AAT ACT GGT TAT GTA G 2057 813-834 20385′-ATC AAG AAT ACT GGC TAC GTA G  2052^(c) 757-778 20395′-ATC AAA AAC ACT GGC TAT GTA G  2053^(c) 787-808 ^(a)These sequenceswere aligned to derive the corresponding primer. ^(b)The nucleotidepositions refer to the pbpla sequence fragment (SEQ ID NO. 1004).^(c)Sequence from databases.

TABLE 76Strategy for the selection of vanAB-specific amplification primers and vanA- and vanB- specific hybridization probes from van sequences. Accession #734                      759       936                      961SEQ ID NO.: vanA X56895GTAGGCT GCGATATTCA AAGCTCAGC . . . CGGACGAATT GGACTACGCA ATTGAA . . .  1139 vanA M97297GTAGGCT GCGATATTCA AAGCTCAGC . . . CGGACGAATT GGACTACGCA ATTGAA . . .  1141 vanAGTAGGCT GCGATATTCA AAGCTCAGC . . . CGGACGAATT GGACTACGCA ATTGAA . . .  1051 vanAGTAGGCT GCGATATTCA AAGCTCAGC . . . CGGACGAATT GGACTACGCA ATTGAA . . .  1052 vanAGTAGGCT GCGATATTCA AAGCTCAGC . . . CGGACGAATT GGACTACGCA ATTGAA . . .  1053 vanAGTAGGCT GCGATATTCA AAGCTCAGC . . . CGGACGAATT GGACTACGCA ATTGAA . . .  1054 vanAGTAGGCT GCGATATTCA AAGCTCAGC . . . CGGACGAATT GGACTACGCA ATTGAA . . .  1055 vanAGTAGGCT GCGATATTCA AAGCTCAGC . . . CGGACGAATT GGACTACGCA ATTGAA . . .  1056 vanAGTAGGCT GCGATATTCA AAGCTCAGC . . . CGGACGAATT GGACTACGCA ATTGAA . . .  1057 vanAGTAGGCT GCGATATTCA AAGCTCAGC . . . CGGACGAATT GGACTACGCA ATTGAA . . .  1049 vanAGTAGGCT GCGATATTCA AAGCTCAGC . . . CGGACGAATT GGACTACGCA ATTGAA . . .  1050 vanB U94526GTGGGCT GTGATATTCA AAGCTCCGC . . . CGGAaGAAcT taACgctGCg ATaGAA . . .  1117 vanB U94527GTAGGCT GCGATATTCA AAGCTCCGC . . . CGGAaGAAcT aaACgctGCg ATaGAA . . . -2594 vanB U94528GTGGGCT GTGATATTCA AAGCTCCGC . . . CGGAaGAAcT taACgctGCg ATaGAA . . . -2595 vanB U94529GTGGGCT GTGATATTCA AAGCTCCGC . . . CGGAaGAAcT taACgctGCg ATaGAA . . . -2596 vanB U94530GTGGGCT GTGATATTCA AAGCTCCGC . . . CGGAaGAAcT taACgctGCg ATaGAA . . . -2597 vanB Z83305GTGGGCT GTGATATTCA AAGCTCCGC . . . CGGAaGAAcT taACgctGCg ATaGAA . . . -2598 vanB U81452GTGGGCT GTGATATTCA AAGCTCCGC . . . CGGAaGAAcT taACgctGCg ATaGAA . . . -2599 vanB U35369GTAGGCT GCGATATTCA AAGCTCCGC . . . CGGAaGAAcT aaACgctGCg ATaGAA . . . -2600 vanB U72704GTGGGCT GCGATATTCA AAGCTCCGC . . . CGGAaGAAcT taACgctGCg ATaGAA . . . -2601 vanB L06138GTAGGCT GCGATATTCA AAGCTCCGC . . . CGGAaGAAcT aaACgctGCg ATaGAA . . . -2602 vanB L15304GTGGGCT GTGATATTCA AAGCTCCGC . . . CGGAaGAAcT taACgctGCg ATaGAA . . . -2603 vanB U00456GTAGGCT GCGATATTCA AAGCTCCGC . . . CGGAaGAAcT aaACgctGCg ATaGAA . . . -2604 vanD AF130997GTGGGaT GCGATATTCA AAGCTCCGT . . . CAGAaGAAcT GcAggcaGCA ATcGAA . . . -2605 vanE AF136925GTAGGtT GTGgTATcgg AgctgCAGC . . . AAAgtGAtTT atAtaAaGCA ATaGAC . . . -2606 Selected sequence for    GGCT GYGATATTCA AAGCTC  1112amplification primer Selected sequence for                                      ACGAATT GGACTACGCA ATT (vanA) 1170 hybridization probeThe sequence numbering refers to the Enterococcus faecium vanA gene fragment (SEQ ID NO. 1139). Nucleotides in capitals are identical to the selected sequences or match those sequences. Mismatches are indicated by lower-case letters. Dots indicate gaps in the sequences displayed.“R” “Y” “M” “K” “W” and “S”designate nucleotide positions which are degenerated. “R” standsfor A or G; “Y” stands for C or T; “M” stands for A or C; “K”stands for G or T; “W” stands for A or T; “S” stands for C or G. “I”stands for inosine which is a nucleotide analog thatcan bind to any of the four nucleotides A, C, G or T. Accession #1038                    1063       1103                          1133SEQ ID NO.: vanA X56895GAAACagt GccGcGTTag TTGTtGGC . . . ATT CATCAGGAAG TCGAGCCGGA AAAAGGCT 1139 vanA M97297GAAACagt GccGcgTTag TTGTtGGC . . . ATT CATCAGGAAG TCGAGCCGGA AAAAGGCT 1141 vanAGAAACagt GccGcgTTag TTGTtGGC . . . ATT CATCAGGAAG TCGAGCCGGA AAAAGGCT 1051 vanAGAAACagt GccGcgTTag cTGTtGGC . . . ATT CATCAGGAAG TCGAGCCGGA AAAAGGCT 1052 vanAGAAACagt GccGcgTTag cTGTtGGC . . . ATT CATCAGGAAG TCGAGCCGGA AAAAGGCT 1053 vanAGAAACagt GccGcgTTag TTGTtGGC . . . ATT CATCAGGAAG TCGAGCCGGA AAAAGGCT 1054 vanAGAAACagt GccGcgTTag cTGTtGGC . . . ATT CATCAGGAAG TCGAGCCGGA AAAAGGCT 1055 vanAGAAACagt GccGcgTTag cTGTtGGC . . . ATT CATCAGGAAG TCGAGCCGGA AAAAGGCT 1056 vanAGAAACagt GccGcgTTag cTGTtGGC . . . ATT CATCAGGAAG TCGAGCCGGA AAAAGGCT 1057 vanAGAAACagt GccGcgTTag cTGTtGGC . . . ATT CATCAGGAAG TCGAGCCGGA AAAAGGCT 1049 vanAGAAACagt GccGcgTTag cTGTtGGC . . . ATT CATCAGGAAG TCGAGCCGGA AAAAGGCT 1050 vanB U94526GGAACGAG GATGATTTGA TTGTCGGC . . . ATC CATCAGGAAA ACGAGCCGGA AAAAGGCT 1117 vanB U94527GAAACGAG GATGATTTGA TTGTCGGC . . . ATC CATCAGGAAA ACGAGCCGGA AAAAGGCT-2607 vanB U94528GGAACGAG GATGATTTGA TTGTCGGC . . . ATC CATCAGGAAA ACGAGCCGGA AAAAGGCT-2608 vanB U94529GGAACGAG GATGATTTGA TTGTCGGC . . . ATC CATCAGGAAA ACGAGCCGGA AAAAGGCT-2609 vanB U94530GGAACGAG GATGATTTGA TTGTCGGC . . . ATC CATCAGGAAA ACGAGCCGGA AAAAGGCT-2610 vanB Z83305GGAACGAG GATGATTTGA TTGTCGGC . . . ATC CATCAGGAAA ACGAGCCGGA AAAAGGCT-2611 vanB U81452GGAACGAG GATGATTTGA TTGTCGGC . . . ATC CATCAGGAAA ACGAGCCGGA AAAAGGCT-2612 vanB U35369GAAACGAG GATGATTTGA TTGTCGGC . . . ATC CATCAGGAAA ACGAGCCGGA AAAAGGCT-2613 vanB U72704GGAACGAG GATGATTTGA TTGTCGGC . . . ATC CATCAGGAAA ACGAGCCGGA AAAAGGAT-2614 vanB L06138GAAACGAG GATGATTTGA TTGTCGGC . . . ATC CATCAGGAAA ACGAGCCGGA AAAAGGCT-2615 vanB L15304GGAACGAG GATGATTTGA TTGTCGGC . . . ATC CATCAGGAAA ACGAGCCGGA AAAAGGCT-2616 vanB U00456GAAACGAG GATGATTTGA TTGTCGGC . . . ATC CATCAGGAAA ACGAGCCGGA AAAAGGCT-2617 vanD AF130997GAAACGga aATGATcTcA TgGctGGC . . . ATT CATCAGGAAG cacAGCCGGA aAAGGGAT-2618 vanE AF136925GGAA...t GAacAaTTGg TcGTtGGA . . . TAT gAagAGaAAt ACaA...... ......TT-2619 Selected sequence for    ACGAG GATGATTTGA TTGTC (vanB)  1171hybridization probe Selected sequence for                                       CATCAGGAAR WCGAGCCGGA AAAAG  1111amplification primers The sequence numbering refers to the Enterococcusfaecium vanA gene fragment (SEQ ID NO. 1139). Nucleotides in capitalsare identical to the selected sequences or match those sequences.Mismatches are indicated by lower-case letters. Dots indicate gaps inthe sequences displayed. “R” and “W” designate nucleotide positionswhich are degenerated. “R” stands for A or G; “W” stands for A or T^(a)This sequence is the reverse-complement of the above selectedprimer.

TABLE 77 Internal hybridization probe for specific detection of mecA.Originating DNA fragment SEQ ID NO. Nucleotide sequence SEQ ID NO.Nucleotide position Resistance gene:        mecA 1177 5′-GCT CAA CAA GTT1178^(a) 1313-1332 CCA GAT TA ^(a)Sequence from databases.

TABLE 78 Specific and ubiquitous primers for nucleic acid amplification(hexA sequences). Originating DNA fragment SEQ ID NO.Nucleotide sequence SEQ ID NO. Nucleotide positionBacterial species:             Streptococcus pneumoniae 11795′-ATT TGG TGA CGG GTG ACT TT 1183^(a) 431-450 1181^(b)5′-AGC AGC TTA CTA GAT GCC GT 1183-1191^(c)  652-671^(d)                               Sequencing primers 11795′-ATT TGG TGA CGG GTG ACT TT 1183^(a) 431-450 1182^(b)5′-AAC TGC AAG AGA TCC TTT GG 1183^(a) 1045-1064 ^(a)Sequences fromdatabases. ^(b)These sequences are from the complementary DNA strand ofthe sequence of the originating fragment given in the Sequence Listing.^(c)These sequences were aligned to derive the corresponding primer.^(d)The nucleotide positions refer to the hexA sequence fragment (SEQ IDNO. 1183).

TABLE 79 Internal hybridization probe for specific detection of hexAsequences. Originating DNA fragment SEQ ID NO. Nucleotide sequenceSEQ ID NO. Nucleotide positionBacterial species:      Streptococcus pneumoniae 1180^(a)5′-TCC ACC GTT GCC AAT CGC A 1183-1191^(b) 629-647^(c) ^(a)Thissequences is from the complementary DNA strand of the sequence of theoriginating fragment given in the Sequence Listing. ^(b)These sequenceswere aligned to derive the corresponding primer. ^(c)The nucleotidepositions refer to the hexA sequence fragment (SEQ ID NO. 1183).

TABLE 80Strategy for the selection of Streptococcus pneumoniae species-specific amplificationprimers and hybridization probe from hexA sequences.428                       453       626                                                SEQ ID NO.: S. pneumoniaeTGG ATTTGGTGAC GGGTGACTTT TAT . . . ATTTG CGATTGGCAA CGGTGGAGCA  1183S. pneumoniae ~~~~~~~~~TGAC GGGTGACTTT TAT . . . ATTTG CGATTGGCAA CGGTGGAGCA  1184S. pneumoniae ~~~~~~~~~TGAC GGGTGACTTT TAT . . . ATTTG CGATTGGCAA CGGTGGAGCA  1185S. pneumoniae ~~~~~~~~~TGAC GGGTGACTTT TAT . . . ATTTG CGATTGGCAA CGGTGGAGCA  1186S. pneumoniae ~~~~~~~~~TGAC GGGTGACTTT TAT . . . ATTTG CGATTGGCAA CGGTGGAGCA  1187S. oralis  ~~~ ~~~~~~~~~~GGGTGACTTT TAT . . . ATCca CGAcTGGCAg CtGTGGAGCA  1188 S. mitis ~~~~~~~GGTGAC GGGTGACTTT TAT . . . ATTca CGATTGGCAg CtGTGGAGCA  1189S. mitis ~~~~~~~~~TGAC GGGTGACTTT CAG . . . GCGaG gagcTGtCtc CtaTGGAGCG  1190S. mitis ~~~~~~~~~TGAC GGGTGACTTT CAG . . . GCGaG gaAcTGtCtc CtaTGGAGCG  1191Selected sequence for     ATTTGGTGAC GGGTGACTTT 1179amplification primer Selected sequences for 1181amplification primers^(a) 1182 Selected sequence for                                       TG CGATTGGCAA CGGTGGA 1180hybridization probe^(a)                       674       1042                    1067 SEQ ID NO.: S. pneumoniaeAACGGCATCT AGTAAGCTGC TCCA . . . AATCCAAAG GATCTCTTGC AGTTGGC 1183S. pneumoniae AACGGCATCT AGTAAGCTGC TCCA . . . AATCCAAAG GATCTCTTG~~~~~~~~ 1184 S. pneumoniaeAACGGCATCT AGTAAGCTGC TCCA . . . AATCCAAAG GATCTCT~~~ ~~~~~~~ 1185S. pneumoniae AACGGCATCT AGTAAGCTGC TCCA . . . AATCCAAAG GATCTCTT~~~~~~~~~ 1186 S. pneumoniaeAACGGCATCT AGTAAGCTGC TCCG . . . AATCCAAAG GATCTCTT~~ ~~~~~~~ 1187S. oralis  AgCGGCAgCT AGTAAGCTcC TCCA . . . ~~~~~~~~~~ ~~~~~~~~~~~~~~~~~ 1188 S. mitisAgCGGCATCT AGTAAaCTGC TTCA . . . AATCCAAAG GATCTCTT~~ ~~~~~~~ 1189S. mitis TcaGGCAgCa gGgAAaCTGC TGGA . . . ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~1190 S. mitis TcaGGCAgCg gGgAAatTGC TAGA . . . AATCCAAAG GATCTCTT~~~~~~~~~ 1191 Selected sequence for 1179 amplification primerSelected sequences for ACGGCATCT  AGTAAGCTGC T 1181amplification primers^(a)                                    CCAAAG GATCTCTTGC AGTT 1182Selected sequence for 1180 hybridization probe^(a) The sequencenumbering refers to the Streptococcus pneumoniae hexA gene fragment (SEQID NO. 1183). Nucleotides in capitals are identical to the selectedsequences or match those sequences. Mismatches are indicated bylower-case letters. Dots indicate gaps in the sequences displayed. “~”indicate incomplete sequence data. ^(a)This sequence is thereverse-complement of the selected primer.

TABLE 81 Specific and ubiquitous primers for nucleic acid amplification(pcp sequence). Originating DNA fragment SEQ ID NO. Nucleotide sequenceSEQ ID NO. Nucleotide positionBacterial species:      Streptococcus pyogenes 12115′-ATT CTT GTA ACA GGC TTT GAT CCC 1215^(a) 291-314 1210^(b)5′-ACC AGC TTG CCC AAT ACA AAG G 1215^(a) 473-494 ^(a)Sequences fromdatabases. ^(b)These sequences are from the complementary DNA strand ofthe sequence of the originating fragment given in the Sequence Listing.

TABLE 82Specific and ubiquitous primers for nucleic acid amplification of S. saprophyticussequences of unknown coding potential. Originating DNA fragmentSEQ ID NO. Nucleotide sequence SEQ ID NO. Nucleotide positionBacterial species:     Staphylococcus saprophyticus 12085′-TCA AAA AGT TTT CTA AAA AAT TTA C 74,1093, 169-193^(c) 1198^(b)1209^(a) 5′-ACG GGC GTC CAC AAA ATC AAT AGG A 74,1093, 355-379^(c)1198^(b) ^(a)This sequence is from the complementary DNA strand of thesequence of the originating fragment given in the Sequence Listing.^(b)These sequences were aligned to derive the corresponding primer.^(c)The nucleotide positions refer to the S. saprophyticus unknown genesequence fragment (SEQ ID NO. 1198).

TABLE 83Molecular beacon internal hybridization probes for specific detection of antimicrobialagents resistance gene sequences. Originating DNA fragment SEQ ID NO.Nucleotide sequence^(a) SEQ ID NO. Nucleotide positionResistance gene:            gyrA 22505′-CCG TCG GAT GGT GTC GTA TAC CGC GGA GTC 1954^(b) 218-243   GCC GAC GG 2251 5′-CGG AGC CGT TCT CGC TGC GTT ACA TGC TGG 1954^(b)259-286    TGG CTC CG Resistance gene:            mecA 12315′-GCG AGC CCG AAG ATA AAA AAG AAC CTC TGC 1178^(b) 1291-1315   TGC TCG C Resistance gene:            parC 1938^(b)5′-CCG CGC ACC ATT GCT TCG TAC ACT GAG GAG 1321^(c) 232-260   TCT CCG CGC GG 1939 5′-CGA CCC GGA TGG TAG TAT CGA TAA TGA TCC1321^(c) 317-346    GCC AGC GGC CGG GTC G 1955^(b)5′-CGC GCA ACC ATT GCT TCG TAC ACT GAG GAG 1321^(c) 235-260   TCT GCG CG Resistance gene:            vanA 12395′-GCG AGC GCA GAC CTT TCA GCA GAG GAG GCT  1051 860-880    CGC 12405′-GCG AGC CGG CAA GAC AAT ATG ACA GCA AAA 1051 663-688    TCG CTC GCResistance gene:            vanB 12415′-GCG AGC GGG GAA CGA GGA TGA TTT GAT TGG 1117 555-577    CTC GCResistance gene:            vanD 15935′-CCG AGC GAT TTA CCG GAT ACT TGG CTG ICG 1594 835-845    CTC GG^(a)Underlined nucleotides indicate the molecular beacon's stem.^(b)This sequence is from the complementary DNA strand of the sequenceof the originating fragment given in the Sequence Listing. ^(c)Sequencefrom databases.

TABLE 84Molecular beacon internal hybridization probe for specific detection ofS. aureus gene sequences of unknown coding potential.Originating DNA fragment SEQ ID NO. Nucleotide sequence^(a) SEQ ID NO.Nucleotide position Bacterial species:           S. aureus 12325′-GGA GCC GCG CGA TTT    1244 53-80    TAT AAA TGA ATG TTG   ATA ACC GGC TCC ^(a)Underlined nucleotides indicate the molecularbeacon's stem.

TABLE 85Molecular beacon internal hybridization probes for specific detection of tuf sequences.Originating DNA fragment SEQ ID NO. Nucleotide sequence^(a) SEQ ID NO.Nucleotide position Bacterial species:          Chlamydia pneumoniae2091 5′-CGC GAC TTG AGA TGG AAC TTA GTG AGC   20 157-183   TTC TTG GTC GCG 2092 5′-CGC GAC GAA AGA ACT TCC TGA AGG TCG   20491-516    TGC AGG TCC AGBacterial species:          Chlamydia trachomatis 22135′-CGT GCC ATT GAC ATG ATT TCC GAA GAA 1739^(b) 412-441   GAC GCT GAA GGC ACG Bacterial species:          Enterococcus faecalis1236 5′-GCG AGC CGT GGT GAA GTT CGC GTT GGT  883 370-391    GGC TCG CBacterial species:          Enterococcus faecium 12355′-GCG AGC CGA AGT TGA AGT TGT TGG TAT   64 412-437    TGC TGG CTC GCBacterial species:          Legionella pneumophila 2084^(c)5′-CAC GCG TCA ACA CCC GTA CAA GTC GTC  112 461-486    TTT TGC GCG TGBacterial species:          Mycoplasma pneumoniae 2096^(c)5′-CGC GAC CGG TAC CAC GGC CAG TAA TCG 2097^(b) 658-679    TGT CGC GBacterial species:          Neisseria gonorrhoeae 21775′-GGC ACG GAC AAA CCA TTC CTG CTG CCT  126 323-357   ATC GAA ACG TGT TCC CGT GCC  21785′-GGC ACG ACA AAC CAT TCC TGC TGC CTA  126 323-348    TCG AAC GTG CC2179 5′-GGC AGC TCT ACT TCC GTA CCA CTG ACG  126 692-718   TAA CCG GCT GCC^(a)Underlined nucleotides indicate the molecular beacon's stem.^(b)Sequence from databases. ^(c)This sequence is from the complementary DNA strand of the sequence of the originating fragment given in the Sequence Listing.Bacterial species:          Pseudomonas aeruginosa 21225′-CCG AGC GAA TGT AGG AGT CCA GGG TCT 153, 880, 280-302^(d)   CTG CTC GG 2138^(b,c)Bacterial species:          Staphylococcus aureus 21865′-ACG CGC TCA AAG CAG AAG TAT ACG TAT 1728 615-646   TAT CAA AAG ACG GCG GT Bacterial group:            Staphylococcus sp. other than S. aureus 12335′-GCG AGC GTT ACT GGT GTA GAA ATG TTC  878 372-394    CGG CTC GCFungal species:             Candida albicans 20735′-CCG AGC AAC ATG ATT GAA CCA TCC ACC  408 404-429    AAC TGG CTC GGFungal species:           Candida dubliniensis 20745′-CCG AGC AAC ATG ATT GAA GCT TCC ACC  414 416-441    AAC TGG CTC GGFungal species:           Candida glabrata 2110^(b)5′-GCG GGC CCT TAA CGA TTT CAG CGA ATC  417 307-335   TGG ATT CAG CCC GC 2111 5′-GCG GGC ATG TTG AAG CCA CCA CCA ACG  417419-447    CTT CCT GGC CCG C Fungal species:           Candida krusei2112^(b) 5′-GCG GGC TTG ATG AAG TTT GGG TTT CCT  422 318-347   TGA CAA TTG CCC GC 2113 5′-GCG GGC ACA AGG GTT GGA CTA AGG AAA  422419-447    CCA AGG CAG CCC GC 21145′-GCG GGC ATC GAT GCT ATT GAA CCA CCT  422 505-533   GTC AGA CCG CCC GC^(a)Underlined nucleotides indicate the molecular beacon's stem.^(b)Sequence from databases.^(c)These sequences were aligned to derive the corresponding primer. ^(d)The nucleotide positions refer to the P. aeruginosa tuf sequence fragment(SEQ ID NO. 153). Fungal species:           Candida lusitaniae 2115^(b)5′-GCG GGC GGT AAG TCC ACC GGT AAG ACC  424 304-330    TTG TTG GCC CGC2116 5′-GCG GGC GTA AGT CAC CGG TAA GAC CTT  424 476-502   GTT GGC CCG C  2117 5′-CGC GGC GAC GCC ATT GAG CCA CCT TCG  424512-535    AGA GCC CGC Fungal species:           Candida parapsilosis2118^(b) 5′-GCG GGC TCC TTG ACA ATT TCT TCG TAT  426 301-330   CTG TTC TTG GCC CGC Fungal species:           Candida tropicalis 21195′-GCG GGC TTA CAA CCC TAA GGC TGT TCC  429 357-384    ATT CGT TGC CGC C2120 5′-GCG GGC AGA AAC CAA GGC TGG TAA GGT  429 459-487   TAC CGG AGC CCG C Fungal species:           Cryptococcus neoformans2106 5′-GCG AGC AGA GCA CGC CCT CCT CGC CGC  623, 1985, 226-244^(d)   TCG C 1986^(c) 2107 5′-GCG AGC TCC CCA TCT CTG GTT GGC ACG 623, 1985, 390-408^(d)    CTC GC 1986^(c)Bacterial genus:          Legionella sp. 20835′-CCG CCG ATG TTC CGT AAA TTA CTT GAI 111-112^(d) 488-519^(e)   GAA GGT CGA GCC GGC GG^(a)Underlined nucleotides indicate the molecular beacon's stem.^(b)This sequence is from the complementary DNA strand of the sequence of the originating fragment given in the Sequence Listing.^(c)These sequences were aligned to derive the corresponding primer. ^(d)The nucleotide positions refer to the C. neoformans tuf (EF-1) sequence fragment(SEQ ID NO. 623).^(e)The nucleotide positions refer to the L. pneumophila tuf (EF-1) sequence fragment(SEQ ID NO. 112). Fungal genus:             Candida sp. 21085′-GCG GGC AAC TTC RTC AAG AAG GTT GGT 414,417, 52-80^(c)   TAC AAC CCG CCC GC 422,424, 426,429,624^(b) 21095′-GCG GGC CCA ATC TCT GGT TGG AAY GGT Same as SEQ 100-125^(c)   GAC AAG CCC GC ID NO. 2108 Bacterial group:          Pseudomonads2121 5′-CGA CCG CIA GCC GCA CAC CAA GTT CCG 153-155, 205, 598-616^(e)   GTC G  880, 2137^(d), 2138^(d,b) ^(a)Underlined nucleotides indicatethe molecular beacon's stem. ^(b)These sequences were aligned to derivethe corresponding primer. ^(c)The nucleotide positions refer to the C.albicans tuf (EF-1) sequence fragment (SEQ ID NO. 624). ^(d)Sequencefrom databases. ^(e)The nucleotide positions refer to the P. aeruginosatuf sequence fragment (SEQ ID NO. 153).

TABLE 86 Molecular beacon internal hybridizationprobes for specific detection of ddl and mtl gene sequences.  Originating DNA fragment SEQ ID Nucleotide SEQ ID NO.Nucleotide sequence^(a) NO. position Bacterial species: E. faecium (ddl)1237 5′-GCG AGC CGC GAA ATC GAA GTT GCT GTA 1242^(b) 334-359TTA GGG CTC GC Bacterial species: E. faecalis (mtl) 12385′-GCG AGC GGC GTT AAT TTT GGC ACC GAA 1243^(b) 631-656 GAA GAG CTC GC^(a)Underlined nucleotides indicate the molecular beacon's stem.^(b)Sequence from databases.

TABLE 87 Internal hybridization probe for specificdetection of S. aureus sequences of unknown coding potential.Originating DNA fragment SEQ ID Nucleotide SEQ ID NO.Nucleotide sequence NO. positionBacterial species: Staphylococcus aureus 12345′-ACT AAA TAA ACG CTC ATT CG 1244 35-54

TABLE 88 Specific and ubiquitous primers for nucleicacid amplification (antimicrobial agents resistance genes sequences).Originating DNA fragment SEQ ID Nucleotide SEQ ID NO.Nucleotide sequence NO. position Resistance gene: aac(2′)-Ia 13445′-AGC AGC AAC GAT GTT ACG CAG CAG 1348^(a) 163-186 1345^(b)5′-CCC GCC GAG CAT TTC AAC TAT TG 1348^(a) 392-414 13465′-GAT GTT ACG CAG CAG GGC AGT C 1348^(a) 172-193 1347^(b)5′-ACC AAG CAG GTT CGC AGT CAA GTA 1348^(a) 467-490Resistance gene: aac(3′)-Ib 1349 5′-CAG CCG ACC AAT GAG TAT CTT GCC1351^(a) 178-201 1350^(b) 5′-TAA TCA GGG CAG TTG CGA CTC CTA 1351^(a)356-379 Resistance gene: aac(3′)-IIb 13525′-CCA CGC TGA CAG AGC CGC ACC G 1356^(a) 383-404 1353^(b)5′-GGC CAG CTC CCA TCG GAC CCT G 1356^(a) 585-606 13545′-CAC GCT GAC AGA GCC GCA CCG 1356^(a) 384-404 1355^(b)5′-ATG CCG TTG CTG TCG AAA TCC TCG 1356^(a) 606-629Resistance gene: aac(3′)-IVa 1357 5′-GCC CAT CCA TTT GCC TTT GC 1361^(a)295-314 1358^(b) 5′-GCG TAC CAA CTT GCC ATC CTG AAG 1361^(a) 517-5401359 5′-TGC CCC TGC CAC CTC ACT C 1361^(a) 356-374 1360^(b)5′-CGT ACC AAC TTG CCA TCC TGA AGA 1361^(a) 516-539Resistance gene: aac(3′)-VIa 1362 5′-CGC CGC CAT CGC CCA AAG CTG G1366^(a) 285-306 1363^(b) 5′-CGG CAT AAT GGA GCG CGG TGA CTG 1366^(a)551-574 1364 5′-TTT CTC GCC CAC GCA GGA AAA ATC 1366^(a) 502-5251365^(b) 5′-CAT CCT CGA CGA ATA TGC CGC G 1366^(a) 681-702Resistance gene: aac(6′)-Ia 1367 5′-CAA ATA TAC TAA CAG AAG CGT TCA1371^(a) 56-79 1368^(b) 5′-AGG ATC TTG CCA ATA CCT TTA T 1371^(a)269-290 1379 5′-AAA CCT TTG TTT CGG TCT GCT AAT 1371^(a) 153-1761380^(b) 5′-AAG CGA TTC CAA TAA TAC CTT GCT 1371^(a) 320-343Resistance gene: aac(6′)-Ic 1372 5′-GCT TTC GTT GCC TTT GCC GAG GTC1376^(a) 157-180 1373^(b) 5′-CAC CCC TGT TGC TTC GCC CAC TC 1376^(a)304-326 1374 5′-AGA TAT TGG CTT CGC CGC ACC ACA 1376^(a) 104-1271375^(b) 5′-CCC TGT TGC TTC GCC CAC TCC TG 1376^(a) 301-323Resistance gene: ant(3′)-Ia 1377 5′-GCC GTG GGT CGA TGT TTG ATG TTA1381^(a) 100-123 1378^(b) 5′-GCT CGA TGA CGC CAA CTA CCT CTG 1381^(a)221-244 1379 5′-AGC AGC AAC GAT GTT ACG CAG CAG 1381^(a) 127-1501380^(b) 5′-CGC TCG ATG ACG CCA ACT ACC TCT 1381^(a) 222-245Resistance gene: ant(4′)-Ia 1382 5′-TAG ATA TGA TAG GCG GTA AAA AGC1386^(a) 149-172 1383^(b) 5′-CCC AAA TTC GAG TAA GAG GTA TT 1386^(a)386-408 1384 5′-GAT ATG ATA GGC GGT AAA AAG C 1386^(a) 151-172 1385^(b)5′-TCC CAA ATT CGA GTA AGA GGT A 1386^(a) 388-409Resistance gene: aph(3′)-Ia 1387 5′-TTA TGC CTC TTC CGA CCA TCA AGC1391^(a) 233-256 1338^(b) 5′-TAC GCT CGT CAT CAA AAT CAC TCG 1391^(a)488-511 1389 5′-GAA TAA CGG TTT GGT TGA TGC GAG 1391^(a) 468-4911390^(b) 5′-ATG GCA AGA TCC TGG TAT CGG TCT 1391^(a) 669-692Resistance gene: aph(3′)-IIa 1392 5′-TGG GTG GAG AGG CTA TTC GGC TAT1396^(a) 43-66 1393^(b) 5′-CAG TCC CTT CCC GCT TCA GTG AC 1396^(a)250-272 1394 5′-GAC GTT GTC ACT GAA GCG GGA AGG 1396^(a) 244-2671395^(b) 5′-CTT GGT GGT CGA ATG GGC AGG TAG 1396^(a) 386-409Resistance gene: aph(3′)-IIIa 1397 5′-GTG GGA GAA AAT GAA AAC CTA T1401^(a) 103-124 1398^(b) 5′-ATG GAG TGA AAG AGC CTG AT 1401^(a) 355-3741399 5′-ACC TAT GAT GTG GAA CGG GAA AAG 1401^(a) 160-183 1400^(b)5′-CGA TGG AGT GAA AGA GCC TGA TG 1401^(a) 354-376Resistance gene: aph(3′)-VIa 1402 5′-TAT TCA ACA ATT TAT CGG AAA CAG1406^(a) 18-41 1403^(b) 5′-TCA GAG AGC CAA CTC AAC ATT TT 1406^(a)175-197 1404 5′-AAA CAG CGT TTT AGA GCC AAA TAA 1406^(a) 36-59 1405^(b)5′-TTC TCA GAG AGC CAA CTC AAC ATT 1406^(a) 177-200Resistance gene: blaCARB 1407 5′-CCC TGT AAT AGA AAA GCA AGT AGG1411^(a) 351-374 1408^(b) 5′-TTG TCG TAT CCC TCA AAT CAC C 1411^(a)556-577 1409 5′-TGG GAT TAC AAT GGC AAT CAG CG 1411^(a) 205-227 1410^(b)5′-GGG GAA TAG GTC ACA AGA TCT GCT T 1411^(a) 329-353Resistance gene: blaCMY-2 1412 5′-GAG AAA ACG CTC CAG CAG GGC 1416^(a)793-813 1413^(b) 5′-CAT GAG GCT TTC ACT GCG GGG 1416^(a) 975-995 14145′-TAT CGT TAA TCG CAC CAT CAC 1416^(a)  90-110 1415^(b)5′-ATG CAG TAA TGC GGC TTT ATC 1416^(a) 439-459Resistance genes: blaCTX-M-1, blaCTX-M-2 14175′-TGG TTA ACT AYA ATC CSA TTG CGG A 1423^(a) 314-338 1418^(b)5′-ATG CTT TAC CCA GCG TCA GAT T 1423^(a) 583-604Resistance gene: blaCTX-M-1 1419 5′-CGA TGA ATA AGC TGA TTT CTC ACG1423^(a) 410-433 1420^(b) 5′-TGC TTT ACC CAG CGT CAG ATT ACG 1423^(a)580-603 1421 5′-AAT TAG AGC GGC AGT CGG GAG GAA 1423^(a) 116-1391422^(b) 5′-GAA ATC AGC TTA TTC ATC GCC ACG 1423^(a) 405-428Resistance gene: blaCTX-M-2 1424 5′-GTT AAC GGT GAT GGC GAC GCT AC1428^(a) 30-52 1425^(b) 5′-GAA TTA TCG GCG GTG TTA ATC AGC 1428^(a)153-176 1426 5′-CAC GCT CAA TAC CGC CAT TCC A 1428^(a) 510-531 1427^(b)5′-TTA TCG CCC ACT ACC CAT GAT TTC 1428^(a) 687-710Resistance gene: blaIMP 1429 5′-TTT ACG GCT AAA GAT ACT GAA AAG T1433^(a) 205-229 1430^(b) 5′-GTT TAA TAA AAC AAC CAC CGA ATA AT 1433^(a)513-538 1431 5′-TAA TTG ACA CTC CAT TTA CGG CTA A 1433^(a) 191-2151432^(b) 5′-ACC GAA TAA TAT TTT CCT TTC AGG CA 1433^(a) 497-522Resistance gene: blaOXA2 1434 5′-CAC AAT CAA GAC CAA GAT TTG CGA T1438^(a) 319-343 1435^(b) 5′-GAA AGG GCA GCT CGT TAC GAT AGA G 1438^(a)532-556 Resistance gene:bla0XA10 1436 5′-CAG CAT CAA CAT TTA AGA TCC CCA1439^(a) 194-217 1437^(b) 5′-CTC CAC TTG ATT AAC TGC GGA AAT TC 1439^(a)479-504 Resistance gene: blaPER-1 14405′-AGA CCG TTA TCG TAA ACA GGG CTA AG 1442^(a) 281-306 1441^(b)5′-TTT TTT GCT CAA ACT TTT TCA GGA TC 1442^(a) 579-604Resistance gene: blaPER-2 1443 5′-CTT CTG CTC TGC TGA TGC TTG GC1445^(a) 32-54 1444^(b) 5′-GGC GAC CAG GTA TTT TGT AAT ACT GC 1445^(a)304-329 Resistance genes: blaPER-1, blaPER-2 14465′-GGC CTG YGA TTT GTT ATT TGA ACT GGT 1442^(a) 414-440 1447^(b)5′-CGC TST GGT CCT GTG GTG GTT TC 1442^(a) 652-674 14485′-GAT CAG GTG CAR TAT CAA AAC TGG AC 1442^(a) 532-557 1449^(b)5′-AGC WGG TAA CAA YCC TTT TAA CCG CT 1442^(a) 671-696Resistance gene:blaSHV 1883 5′-AGC CGC TTG AGC AAA TTA AAC TA 1900^(a)71-93 1884^(b) 5′-GTA TCC CGC AGA TAA ATC ACC AC 1900^(a) 763-785 18855′-AGC GAA AAA CAC CTT GCC GAC 1900^(a) 313-333 1884^(b)5′-GTA TCC CGC AGA TAA ATC ACC AC 1900^(a) 763-785Resistance gene: blaTEM 1906 5′-CCT TAT TCC CTT TTT TGC GG 1927^(a)27-46 1907^(b) 5′-CAC CTA TCT CAG CGA TCT GTC T 1927^(a) 817-838 19085′-AAC AGC GGT AAG ATC CTT GAG AG 1927^(a) 148-170 1907^(b)5′-CAC CTA TCT CAG CGA TCT GTC T 1927^(a) 817-838 Resistance gene: catI2145 5′-GCA AGA TGT GGC GTG TTA CGG T 2147^(a) 363-384 2146^(b)5′-GGG GCG AAG AAG TTG TCC ATA TT 2147^(a) 484-506Resistance gene: catII 2148 5′-CAG ATT AAA TGC GGA TTC AGC C 2150^(a)67-88 2149^(b) 5′-ATC AGG TAA ATC ATC AGC GGA TA 2150^(a) 151-173Resistance gene: catIII 2151 5′-ATA TTT CAG CAT TAC CTT GGG TT 2153^(a)419-441 2152^(b) 5′-TAC ACA ACT CTT GTA GCC GAT TA 2153^(a) 603-625Resistance gene: catP 2154 5′-CGC CAT TCA GAG TTT AGG AC 2156^(a)178-197 2155^(b) 5′-TTC CAT ACC GTT GCG TAT CAC TT 2156^(a) 339-361Resistance gene: cat 2157 5′-CCA CAG AAA TTG ATA TTA GTG TTT TAT2159^(a)  89-115 2158^(b) 5′-TCG CTA TTG TAA CCA GTT CTA 2159^(a)201-221 2160 5′-TTT TGA ACA CTA TTT TAA CCA GC 2162^(a) 48-70 2161^(b)5′-GAT TTA ACT TAT CCC AAT AAC CT 2162^(a) 231-253 Resistance gene: dfrA1450 5′-ACC ACT GGG AAT ACA CTT GTA ATG GC 1452^(a) 106-131 1451^(b)5′-ATC TAC CTG GTC AAT CAT TGC TTC GT 1452^(a) 296-321Resistance 'gene: dhfrIa 1457 5′-CAA AGG TGA ACA GCT CCT GTT T 1461^(a)75-96 1458^(b) 5′-TCC GTT ATT TTC TTT AGG TTG GTT AAA 1461^(a) 249-2751459 5′-AAG GTG AAC AGC TCC TGT TT 1461^(a) 77-96 1560^(b)5′-GAT CAC TAC GTT CTC ATT GTC A 1461^(a) 207-228Resistance genes: dhfrIa, dhfrXV 14535′-ATC GAA GAA TGG AGT TAT CGG RAA TG 1461^(a) 27-52 1454^(b)5′-CCT AAA AYT RCT GGG GAT TTC WGG A 1461^(a) 384-408 14555′-CAG GTG GTG GGG AGA TAT ACA AAA 1461^(a) 290-313 1456^(b)5′-TAT GTT AGA SRC GAA GTC TTG GKT AA 1461^(a) 416-441Resistance gene: dhfrIb 1466 5′-AAG CAT TGA CCT ACA ATC AGT GT 1470^(a) 98-120 1467^(b) 5′-AAT ACA ACT ACA TTG TCA TCA TTT GAT 1470^(a) 204-2301468 5′-CGT TAC CCG CTC AGG TTG GAC ATC AA 1470^(a) 183-208 1469^(b)5′-CAT CCC CCT CTG GCT CGA TGT CG 1470^(a) 354-376Resistance gene: dhfrV 1471 5′-GAT AAT GAC AAC GTA ATA GTA TTC CC1475^(a) 208-233 1472^(b) 5′-GCT CAA TAT CAA TCG TCG ATA TA 1475^(a)342-364 1473 5′-TTA AAG CCT TGA CGT ACA ACC AGT GG 1475^(a)  95-1201474^(b) 5′-TGG GCA ATG TTT CTC TGT AAA TCT CC 1475^(a) 300-325Resistance genes: dhfrIb, dhfrV 1462 5′-GCA CTC CCY AAT AGG AAA TAC GC1470^(a) 157-179 1463^(b) 5′-AGT GTT GCT CAA AAA CAA CTT CG 1470^(a)405-427 1464 5′-ACG TTY GAA TCT ATG GGM GCA CT 1470^(a) 139-161 1465^(b)5′-GTC GAT AAG TGG AGC GTA GAG GC 1470^(a) 328-350Resistance gene: dhfrVI 1476 5′-GGC GAG CAG CTC CTA TTC AAA G 1480^(a) 79-100 1477^(b) 5′-TAG GTA AGC TAA TGC CGA TTC AAC A 1480^(a) 237-2611478 5′-GAG AAT GGA GTA ATT GGC TCT GGA TT 1480^(a) 31-56 1479^(b)5′-GCG AAA TAC ACA ACA TCA GGG TCA T 1480^(a) 209-233Resistance gene: dhfrVII 1485 5′-AAA ATG GCG TAA TCG GTA ATG GC 1489^(a)32-54 1486^(b) 5′-CAT TTG AGC TTG AAA TTC CTT TCC TC 1489^(a) 189-2141487 5′-AAT CGA AAA TAT GCA GTA GTG TCG AG 1489^(a) 166-191 1488^(b)5′-AGA CTA TTG TAG ATT TGA CCG CCA 1489^(a) 294-317Resistance genes: dhfrVII, dhfrXVII 14815′-RTT ACA GAT CAT KTA TAT GTC TCT 1489^(a) 268-291 1482^(b)5′-TAA TTT ATA TTA GAC AWA AAA AAC TG 1489^(a) 421-446 14835′-CAR YGT CAG AAA ATG GCG TAA TC 1489^(a) 23-45 1484^(b)5′-TKC AAA GCR WTT TCT ATT GAA GGA AA 1489^(a) 229-254Resistance gene: dhfrVIII 1490 5′-GAC CTA TGA GAG CTT GCC CGT CAA A1494^(a) 144-168 1491^(b) 5′-TCG CCT TCG TAC AGT CGC TTA ACA AA 1494^(a)376-401 1492 5′-CAT TTT AGC TGC CAC CGC CAA TGG TT 1494^(a) 18-431493^(b) 5′-GCG TCG CTG ACG TTG TTC ACG AAG A 1494^(a) 245-269Resistance gene: dhfrIX 1495 5′-TCT CTA AAC ATG ATT GTC GCT GTC 1499^(a) 7-30 1496^(b) 5′-CAG TGA GGC AAA AGT TTT TCT ACC 1499^(a) 133-156 14975′-CGG ACG ACT TCA TGT GGT AGT CAG T 1499^(a) 171-195 1498^(b)5′-TTT GTT TTC AGT AAT GGT CGG GAC CT 1499^(a) 446-471Resistance gene: dhfrXII 1500 5′-ATC GGG TTA TTG GCA ATG GTC CTA1504^(a) 50-73 1501^(b) 5′-GCG GTA GTT AGC TTG GCG TGA GAT T 1504^(a)201-225 1502 5′-GCG GGC GGA GCT GAG ATA TAC A 1504^(a) 304-325 1503^(b)5′-AAC GGA GTG GGT GTA CGG AAT TAC AG 1504^(a) 452-477Resistance gene: dhfrXIII 1505 5′-ATT TTT CGC AGG CTC ACC GAG AGC1507^(a) 106-129 1506^(b) 5′-CGG ATG AGA CAA CCT CGA ATT CTG CTG1507^(a) 413-439 Resistance gene: dhfrXV 15085′-AGA ATG TAT TGG TAT TTC CAT CTA TCG 1512^(a) 215-241 1509^(b)5′-CAA TGT CGA TTG TTG AAA TAT GTA AA 1512^(a) 336-361 15105′-TGG AGT GCC AAA GGG GAA CAA T 1512^(a) 67-88 1511^(b)5′-CAG ACA CAA TCA CAT GAT CCG TTA TCG 1512^(a) 266-292Resistance gene: dhfrXVII 1513 5′-TTC AAG CTC AAA TGA AAA CGT CC1517^(a) 201-223 1514^(b) 5′-GAA ATT CTC AGG CAT TAT AGG GAA T 1517^(a)381-405 1515 5′-GTG GTC AGT AAA AGG TGA GCA AC 1517^(a) 66-88 1516^(b)5′-TCT TTC AAA GCA TTT TCT ATT GAA GG 1517^(a) 232-257Resistance gene: embB 2102 5′-CAC CTT CAC CCT GAC CGA CG 2105^(a)822-841 2103^(b) 5′-CGA ACC AGC GGA AAT AGT TGG AC 2105^(a) 948-970Resistance genes: ereA, ereA2 1528 5′-AAC TTG AGC GAT TTT CGG ATA CCC TG1530^(a)  80-105 1529^(b) 5′-TTG CCG ATG AAA TAA CCG CCG ACT 1530^(a)317-340 Resistance gene: ereB 1531 5′-TCT TTT TGT TAC GAC ATA CGC TTT T1535^(a) 152-176 1532^(b) 5′-AGT GCT TCT TTA TCC GCT GTT CTA 1535^(a)456-479 1533 5′-CAG CGG ATA AAG AAG CAC TAC ACA TT 1535^(a) 461-4861534^(b) 5′-CCT CCT GAA ATA AAG CCC GAC AT 1535^(a) 727-749Resistance gene: gyrA 1340 5′-GAA CAA GGT ATG ACA CCG GAT AAA T 1299^(a)163-188 1341^(b) 5′-GAT AAC TGA AAT CCT GAG CCA TAC G 1299^(a) 274-2991936 5′-TAC CAC CCG CAC GGC 1954^(a) 205-219 1937^(b)5′-CGG AGT CGC CGT CGA TG 1954^(a) 309-325 19425′-GAC TGG AAC AAA GCC TAT AAA AAA TCA 1954^(a) 148-174 1937^(b)5′-CGG AGT CGC CGT CGA TG 1954^(a) 309-325 20405′-TGT GAC CCC AGA CAA ACC C 2054^(a) 33-51 2041^(b)5′-GTT GAG CGG CAG CAC TAT CT 2054^(a) 207-226 Resistance gene: inhA2098 5′-CTG AGT CAC ACC GAC AAA CGT C 2101^(a) 910-931 2099^(b)5′-CCA GGA CTG AAC GGG ATA CGA A 2101^(a) 1074-1095Resistance genes: linA, linA′ 1536 5′-AGA TGT ATT AAC TGG AAA ACA ACA A1540^(a)  99-123 1537^(b) 5′-CTT TGT AAT TAG TTT CTG AAA ACC A 1540^(a)352-376 1538 5′-TTA GAA GAT ATA GGA TAC AAA ATA GAA G  1540^(a) 187-2141539^(b) 5′-GAA TGA AAA AGA AGT TGA GCT T 1540^(a) 404-425Resistance gene: linB 1541 5′-TGA TAA TCT TAT ACG TGG GGA ATT T 1545^(a)246-270 1542^(b) 5′-ATA ATT TTC TAA TTG CCC TGT TTC AT 1545^(a) 359-3841543 5′-GGG CAA TTA GAA AAT TAT TTA TCA GA 1545^(a) 367-392 1544^(b)5′-TTT TAC TCA TGT TTA GCC AAT TAT CA 1545^(a) 579-604Resistance gene: mefA 1546 5′-CAA GAA GGA ATG GCT GTA CTA C 1548^(a)625-646 1547^(b) 5′-TAA TTC CCA AAT AAC CCT AAT AAT AGA 1548^(a) 816-842Resistance gene: mefE 1549 5′-GCT TAT TAT TAG GAA GAT TAG GGG GC1551^(a) 815-840 1550^(b) 5′-TAG CAA GTG ACA TGA TAC TTC CGA 1551^(a)1052-1075 Resistance genes: mefA, mefE 15525′-GGC AAG CAG TAT CAT TAA TCA CTA 1548^(a) 50-73 1553^(b)5′-CAA TGC TAC GGA TAA ACA ATA CTA TC 1548^(a) 318-343 15545′-AGA AAA TTA AGC CTG AAT ATT TAG GAC 1548^(a) 1010-1035 1555^(b)5′-TAG TAA AAA CCA ATG ATT TAC ACC G 1548^(a) 1119-1143Resistance genes: mphA, mphK 1556 5′-ACT GTA CGC ACT TGC AGC CCG ACA T1560^(a) 33-57 1557^(b) 5′-GAA CGG CAG GCG ATT CTT GAG CAT 1560^(a)214-237 1558 5′-GTG GTG GTG CAT GGC GAT CTC T 1560^(a) 583-604 1559^(b)5′-GCC GCA GCG AGG TAC TCT TCG TTA 1560^(a) 855-878Resistance gene: mupA 2142 5′-GCC TTA ATT TCG GAT AGT GC 2144^(a)1831-1850 2143^(b) 5′-GAG AAA GAG CCC AAT TAT CTA ATG T 2144^(a)2002-2026 Resistance gene: parC 1342 5′-GAT GTT ATT GGT CAA TAT CAT CCA1321^(a) 205-229 1343^(b) 5′-AAG AAA CTG TCT CTT TAT TAA TAT CAC GT 1321^(a) 396-425 1934 5′-GAA CGC CAG CGC GAA ATT CAA AAA G 1781  67-911935^(b) 5′-AGC TCG GCA TAC TTC GAC AGG 1781  277-297 20445′-ACC GTA AGT CGG CCA AGT CA 2055^(a) 176-195 2045^(b)5′-GTT CTT TCT CCG TAT CGT C 2055^(a) 436-454Resistance gene: ppflo-like 2163 5′-ACC TTC ATC CTA CCG ATG TGG GTT2165^(a) 922-945 2164^(b) 5′-CAA CGA CAC CAG CAC TGC CAT TG 2165^(a)1136-1158 Resistance gene: rpoB 2065 5′-CCA GGA CGT GGA GGC GAT CAC A2072^(a) 1218-1239 2066^(b) 5′-CAC CGA CAG CGA GCC GAT CAG A 2072^(a)1485-1506 Resistance gene: satG 15815′-AAT TGG GGA CTA CAC CTA TTA TGA TG 1585^(a)  93-118 1582^(b)5′-GGC AAA TCA GTC AGT TCA GGA GT 1585^(a) 310-332 15835′-CGA TTG GCA ACA ATA CAC TCC TG 1585^(a) 294-316 1584^(b)5′-TCA CCT ATT TTT ACG CCT GGT AGG AC 1585^(a) 388-413Resistance gene: sulII 1961 5′-GCT CAA GGC AGA TGG CAT TCC C 1965^(a)222-243 1962^(b) 5′-GGA CAA GGC GGT TGC GTT TGA T 1965^(a) 496-517 19635′-CAT TCC CGT CTC GCT CGA CAG T 1965^(a) 237-258 1964^(b)5′-ATC TGC CTG CCC GTC TTG C 1965^(a) 393-411 Resistance gene: tetB 19665′-CAT GCC AGT CTT GCC AAC G 1970^(a) 66-84 1967^(b)5′-CAG CAA TAA GTA ATC CAG CGA TG 1970^(a) 242-264 19685′-GGA GAG ATT TCA CCG CAT AG 1970^(a) 457-476 1969^(b)5′-AGC CAA CCA TCA TGC TAT TCC A 1970^(a) 721-742 Resistance gene: tetM1586 5′-ATT CCC ACA ATC TTT TTT ATC AAT AA 1590^(a) 361-386 1587^(b)5′-CAT TGT TCA GAT TCG GTA AAG TTC 1590^(a) 501-524 15885′-GTT TTT GAA GTT AAA TAG TGT TCT T 1590^(a) 957-981 1589^(b)5′-CTT CCA TTT GTA CTT TCC CTA 1590^(a) 1172-1192 Resistance gene: vatB1609 5′-GCC CTG ATC CAA ATA GCA TAT A 1613^(a) 11-32 1610^(b)5′-CCT GGC ATA ACA GTA ACA TTC TG 1613^(a) 379-401 16115′-TGG GAA AAA GCA ACT CCA TCT C 1613^(a) 301-322 1612^(b)5′-ACA ACT GAA TTC GCA GCA ACA AT 1613^(a) 424-446 Resistance gene: vatC1614 5′-CCA ATC CAG AAG AAA TAT ACC C 1618^(a) 26-47 1615^(b)5′-ATT AGT TTA TCC CCA ATC AAT TCA 1618^(a) 177-200 16165′-ATA ATG AAT GGG GCT AAT CAT CGT AT 1618^(a) 241-266 1617^(b)5′-GCC AAC AAC TGA ATA AGG ATC AAC 1618^(a) 463-486 Resistance gene: vga1619 5′-AAG GCA AAA TAA AAG GAG CAA AGC 1623^(a) 641-664 1620^(b)5′-TGT ACC CGA GAC ATC TTC ACC AC 1623^(a) 821-843 16215′-AAT TGA AGG ACG GGT ATT GTG GAA AG 1623^(a) 843-868 1622^(b)5′-CGA TTT TGA CAG ATG GCG ATA ATG AA 1623^(a)  975-1000Resistance gene: vgaB 1624 5′-TTC TTT AAT GCT CGT AGA TGA ACC TA1628^(a) 354-379 1625^(b) 5′-TTT TCG TAT TCT TCT TGT TGC TTT C 1628^(a)578-602 1626 5′-AGG AAT GAT TAA GCC CCC TTC AAA AA 1628^(a) 663-6881627^(b) 5′-TTA CAT TGC GAC CAT GAA ATT GCT CT 1628^(a) 849-874Resistance genes: vgb, vgh 1629 5′-AAG GGG AAA GTT TGG ATT ACA CAA CA1633^(a) 73-98 1630^(b) 5′-GAA CCA CAG GGC ATT ATC AGA ACC 1633^(a)445-468 1631 5′-CGA CGA TGC TTT ATG GTT TGT 1633^(a) 576-596 1632^(b)5′-GTT AAT TTG CCT ATC TTG TCA CAC TC 1633^(a) 850-875Resistance gene: vgbB 1634 5′-TTA ACT TGT CTA TTC CCG ATT CAG G 1882^(a)23-47 1635^(b) 5′-GCT GTG GCA ATG GAT ATT CTG TA 1882^(a) 267-289 16365′-TTC CTA CCC CTG ATG CTA AAG TGA 1882^(a) 155-178 1637^(b)5′-CAA AGT GCG TTA TCC GAA CCT AA 1882^(a) 442-464 Sequencing primersResistance gene: gyrA 1290 5′-GAY TAY GCI ATG ISI GTI ATH GT 1299^(a)70-83 1292^(b) 5′-ARI SCY TCI ARI ATR TGI GC 1299^(a) 1132-1152 12915′-GCI YTI CCI GAY GTI MGI GAY GG 1299^(a) 100-123 1292^(b)5′-ARI SCY TCI ARI ATR TGI GC 1299^(a) 1132-1152 12935′-ATG GCT GAA TTA CCT CAA TC 1299^(a)  1-21 1294^(b)5′-ATG ATT GTT GTA TAT CTT CTT CAA C 1299^(a) 2626-2651 1295^(b)5′-CAG AAA GTT TGA AGC GTT GT 1299^(a) 1255-1275 12965′-AAC GAT TCG TGA GTC AGA TA 1299^(a) 1188-1208 12975′-CGG TCA ACA TTG AGG AAG AGC T 1300^(a) 29-51 1298^(b)5′-ACG AAA TCG ACC GTC TCT TTT TC 1300^(a) 415-437 Resistance gene: gyrB1301 5′-GTI MGI AWI MGI CCI GSI ATG TA 1307^(a)  82-105 1302^(b)5′-TAI ADI GGI GGI KKI GCI ATR TA 1307^(a) 1600-1623 13035′-GGI GAI GAI DYI MGI GAR GG 1307^(a) 955-975 1304^(b)5′-CIA RYT TIK YIT TIG TYT G 1307^(a) 1024-1043 13055′-ATG GTG ACT GCA TTG TCA GAT G 1307^(a)  1-23 1306^(b)5′-GTC TAC GGT TTT CTA CAA CGT C 1307^(a) 1858-1888Resistance gene: parC 1308 5′-ATG TAY GTI ATI ATG GAY MGI GC 1320^(a)67-90 1309^(b) 5′-ATI ATY TTR TTI CCY TTI CCY TT 1320^(a) 1993-2016 13105′-ATI ATI TSI ATI ACY TCR TC 1320^(a) 1112-1132 1311^(b)5′-GAR ATG AAR ATI MGI GGI GAR CA 1320^(a) 1288-1311 13125′-AAR TAY ATI ATI CAR GAR MGI GC 1321^(a) 67-90 1313^(b)5′-AMI AYI CKR TGI GGI TTI TTY TT 1321^(a) 2212-2235 13145′-TAI GAI TTY ACI GAI SMI CAR GC 1321^(a) 1228-1251 1315^(b)5′-ACI ATI GCI TCI GCY TGI KSY TC 1321^(a) 1240-1263 13165′-GTG AGT GAA ATA ATT CAA GAT T 1321^(a)  1-23 1317^(b)5′-CAC CAA AAT CAT CTG TAT CTA C 1321^(a) 2356-2378 13185′-ACC TAY TCS ATG TAC GTR ATC ATG GA  1320^(a) 58-84 1319^(b)5′-AGR TCG TCI ACC ATC GGY AGY TT 1320^(a) 832-855 Resistance gene: parE1322 5′-RTI GAI AAY ISI GTI GAY GAR G 1328^(a) 133-155 1325^(b)5′-RTT CAT YTC ICC IAR ICC YTT 1328^(a) 1732-1752 13235′-ACI AWR SAI GGI GGI ACI CAY G 1328^(a) 829-850 1324^(b)5′-CCI CCI GCI SWR TCI CCY TC 1328^(a) 1280-1302 13265′-TGA TTC AAT ACA GGT TTT AGA G 1328^(a) 27-49 1327^(b)5′-CTA GAT TTC CTC CTC ATC AAA T 1328^(a) 1971-1993 ^(a)Sequence fromdatabases. ^(b)These sequences are from the complementary DNA strand ofthe sequence of the originating fragment given in the Sequence Listing.

TABLE 89 Internal hybridization probes for specificdetection of antimicrobial agents resistance genes sequences.Originating DNA fragment SEQ ID Nucleotide SEQ ID NO.Nucleotide sequence NO. position Resistance gene: aph3'VIa 22525′-CCA CAT ACA GTG TCT CTC 1406^(a) 149-166 Resistance gene:blaSHV 18865′-GAC GCC CGC GCC ACC ACT 1900^(a) 484-501 18875′-GAC GCC CGC GAC ACC ACT A 1899^(a) 514-532 18885′-GAC GCC CGC AAC ACC ACT A 1901^(a) 514-532 18895′-GTT CGC AAC TGC AGC TGC TG 1899^(a) 593-612 18905′-TTC GCA ACG GCA GCT GCT G 1899^(a) 594-612 18915′-CCG GAG CTG CCG AIC GGG 1902^(a) 692-709 18925′-CGG AGC TGC CAA RCG GGG 1903^(a) 693-710 18935′-GGA GCT GGC GAR CGG GGT 1899^(a) 694-711 18945′-GAC CGG AGC TAG CGA RCG 1904^(a) 690-707 18955′-CGG AGC TAG CAA RCG GGG T 1905^(a) 693-711 18965′-GAA ACG GAA CTG AAT GAG GCG 1899^(a) 484-504 18975′-CAT TAC CAT GGG CGA TAA CAG 1899^(a) 366-386 18985′-CCA TTA CCA TGA GCG ATA ACAG 1899^(a) 365-386 Resistance gene: blaTEM1909 5′-ATG ACT TGG TTA AGT ACT CAC C 1928^(a) 293-314 19105′-ATG ACT TGG TTG AGT ACT CAC C 1927^(a) 293-314 19115′-CCA TAA CCA TGG GTG ATA ACA C 1928^(a) 371-392 19125′-CCA TAA CCA TGA GTG ATA ACA C 1927^(a) 371-392 19135′-CGC CTT GAT CAT TGG GAA CC 1928^(a) 475-494 19145′-CGC CTT GAT CGT TGG GAA CC 1927^(a) 475-494 19155′-CGC CTT GAT AGT TGG GAA CC 1929^(a) 475-494 19165′-CGT GGG TCT TGC GGT ATC AT 1927^(a) 712-731 19175′-CGT GGG TCT GGC GGT ATC AT 1930^(a) 712-731 19185′-GTG GGT CTC ACG GTA TCA TTG 1927^(a) 713-733 19195′-CGT GGG TCT CTC GGT ATC ATT 1931^(a) 712-732 19205′-CGT GGI TCT CGC GGT ATC AT 1927^(a) 712-731 19215′-CGT GGG TCT AGC GGT ATC ATT 1932^(a) 713-733 19225′-GTT TTC CAA TGA TTA GCA CTT TTA 1927^(a) 188-211 19235′-GTT TTC CAA TGA TAA GCA CTT TTA 1927^(a) 188-211 19245′-GTT TTC CAA TGC TGA GCA CTT TT 1932^(a) 188-210 19255′-CGT TTT CCA ATG ATG AGC ACT TT 1927^(a) 187-209 19265′-GTT TTC CAA TGG TGA GCA CTT TT 1933^(a) 188-210 20065′-TGG AGC CGG TGA GCG TGG 1927^(a) 699-716 20075′-TGG AGC CAG TGA GCG TGG 2010^(a) 699-716 20085′-TCT GGA GCC GAT GAG CGT G 1929^(a) 697-715 20095′-CTG GAG CCA GTA AGC GTG G 2011^(a) 698-716 21415′-CAC CAG TCA CAG AAA AGC 1927^(a) 311-328 Resistance gene: dhfrIa 22535′-CAT TAC CCA ACC GAA AGT A 1461^(a) 158-176 Resistance gene: embB 21045′-CTG GGC ATG GCI CGA GTC 2105^(a) 910-927 Resistance gene: gyrA 13335′-TCA TGG TGA CTT ATC TAT TTA TG 1299^(a) 240-263 13345′-CAT CTA TTT ATA AAG CAA TGG TA 1299^(a) 251-274 13355′-CTA TTT ATG GAG CAA TGG T 1299^(a) 254-273 19405′-GTA TCG TTG GTG ACG TAA T 1299^(a) 206-224 19435′-GCT GGT GGA CGG CCA G 1954^(a) 279-294 1945 5′-CGG CGA CTA CGC GGT AT1954^(a) 216-232 1946 5′-CGG CGA CTT CGC GGT AT 1954^(a) 216-232 19475′-CGG TAT ACG GCA CCA TCG T 1954^(a) 227-245 19485′-GCG GTA TAC AAC ACC ATC G 1954^(a) 226-244 19495′-CGG TAT ACG CCA CCA TCG T 1954^(a) 227-245 20425′-CAC GGG GAT TTC TCT ATT TA 2054^(a) 103-122 20435′-CAC GGG GAT TAC TCT ATT TA 2054^(a) 103-122 Resistance gene: inhA2100 5′-GCG AGA CGA TAG GTT GTC 2101^(a) 1017-1034 Resistance gene: parC1336 5′-TGG AGA CTA CTC AGT GT 1321^(a) 232-249 13375′-TGG AGA CTT CTC AGT GT 1321^(a) 232-249 1338 5′-GTG TAC GGA GCA ATG1321^(a) 245-260 1339 5′-CCA GCG GAA ATG CGT 1321^(a) 342-357 19415′-GCA ATG GTC CGT TTA AGT 1321^(a) 253-270 19445′-TTT CGC CGC CAT GCG TTA C 1781  247-265 1950 5′-GGC GAC ATC GCC TGC1781  137-151 1951 5′-GGC GAC AGA GCC TGC TA 1781  137-153 19525′-CCT GCT ATG GAG CGA TGG T 1781  147-165 19535′-CGC CTG CTA TAA AGC GAT GGT 1781  145-165 20465′-ACG GGG ATT TTT CTA TCT AT 2055^(a) 227-246 Resistance gene: rpoB2067 5′-AGC TGA GCC AAT TCA TGG 2072^(a) 1304-1321 20685′-ATT CAT GGA CCA GAA CAA C 2072^(a) 1314-1332 20695′-CGC TGT CGG GGT TGA CCC 2072^(a) 1334-1351 20705′-GTT GAC CCA CAA GCG CCG 2072^(a) 1344-1361 20715′-CGA CTG TCG GCG CTG GGG 2072^(a) 1360-1377 Resistance gene: tetM 22545′-ACC TGA ACA GAG AGA AAT G 1590^(a) 1062-1080 ^(a)Sequence fromdatabases.

TABLE 90 Molecular beacon internal hybridizationprobes for specific detection of atpD sequences. OriginatingDNA fragment SEQ ID Nucleotide SEQ ID NO. Nucleotide sequence^(a) NO.position Bacterial species: Bacteroides fragilis 21365′-CCA ACG CGT CCT CAA TCA TTT CTA ACT TCT 929 353-382ATG GCC GGC GTT GG Bacterial species: Bordetella pertussis 21825′-GCG CGC CAA CGA CTT CTA CCA CGA AAT GGA 1672 576-605 AGA GTC GCG CGCBacterial group: Campylobacter jejuni and C. coli 21335′-CCA CGC ACA WAA ACT TGT TTT AGA AGT 1576, 44-73^(d)AGC AGC WCA GCG TGG 1600,1849, 1863,2139^(b,c)Fungal species: Candida glabrata 20785′-CCG AGC CTT GGT CTT CGG CCA AAT GAA CGC 463 442-463 TCG GFungal species: Candida krusei 20755′-CCG AGC CAG GTT CTG AAG TCT CTG CAT TAT 468 720-748 TAG GTG CTC GGFungal species: Candida lusitaniae 20805′-CCG AGC CGA AGA GGG CCA AGA TGT CGC TCG 470 520-538 GFungal species: Candida parapsilosis 20795′-CCG AGC GTT CAG TTA CTT CAG TCC AAG CCG 472 837-860 GCT CGGFungal species: Candida tropicalis 20775′-CCG AGC AAC CGA TCC AGC TCC AGC TAC GCT 475 877-897 CGGBacterial species: Klebsiella pneumoniae 22815′-CCC CCA GCT GGG CGG CGG TAT CGA TGG GGG 317 40-59^(a)Underlined nucleotides indicate the molecular beacon's stem.^(b)Sequence from databases.^(c)These sequences were aligned to derive the corresponding primer.^(d)The nucleotide positions refer to the C. jejuni atpD sequence fragment (SEQID NO. 1576). Fungal genus: Candida sp. 20765′-CCG AGC YGA YAA CAT TTT CAG ATT CAC CCA  460-478, 697-723^(c)RGC GCT CGG 663^(b)^(a)Underlined nucleotides indicate the molecular beacon's stem.^(b)These sequences were aligned to derive the corresponding primer.^(c)The nucleotide positions refer to the C. albicans atpD sequence fragment(SEQ ID NO. 460).

TABLE 91 Internal hybridization probes for specificdetection of atpD sequences. Originating  DNA fragment SEQ ID NucleotideSEQ ID NO. Nucleotide sequence NO. positionBacterial species: Acinetobacter baumannii 21695′-CCC GTT TGC GAA AGG TGG 243 304-321Bacterial species: Klebsiella pneumoniae 2167 5′-CAG CAG CTG GGC GGC GGT317 36-53

TABLE 92 Internal hybridization probes for specificdetection of ddl and mtl sequences. Originating DNA fragment SEQ IDNucleotide SEQ ID NO. Nucleotide sequence NO. positionBacterial species: Enterococcus faecium (ddl) 22865′-AGT TGC TGT ATT AGG AAA TG 2288^(a) 784-803 22875′-TCG AAG TTG CTG TAT TAG GA 2288^(a) 780-799Bacterial species: Enterococcus faecalis mtl) 22895′-CAC CGA AGA AGA TGA AAA AA 1243^(a) 264-283 22905′-TGG CAC CGA AGA AGA TGA 1243^(a) 261-278 22915′-ATT TTG GCA CCG AAG AAG A 1243^(a) 257-275 ^(a)Sequence fromdatabases.

1. A composition for the detection of Klebsiella pneumoniae in a sampleusing a nucleic acid amplification assay, comprising an amplificationprimer pair, said amplification primer pair consisting of anoligonucleotide consisting of SEQ ID NO: 1331 or the complement thereof,and an oligonucleotide consisting of SEQ ID NO: 1332 or the complementthereof, or variants of either SEQ ID NO: 1331 or 1332, or both, whereinsaid variants differ from SEQ ID NO: 1331 or 1332 in that they have upto three nucleotide changes compared to SEQ ID NO:1331 or 1332, whereinsaid variants are capable of hybridizing to and amplifying K. pneumoniaenucleic acids in said nucleic acid amplification assay and wherein eacholigonucleotide optionally includes a detectable moiety.
 2. Thecomposition of claim 1, further comprising a probe that hybridizes to aportion of the atpD gene amplified by said amplification primer pair. 3.The composition of claim 2, wherein said probe comprises a fluorescentmoiety.
 4. The composition of claim 2, wherein said probe is a molecularbeacon.
 5. A method of detecting Klebsiella pneumoniae in a samplecomprising: a) contacting the sample with the composition according toclaim 1; b) amplifying target nucleic acid in the sample of a) togenerate amplification product(s); and c) detecting the presence oramount of amplification product(s) as an indication of the presence ofthe Klebsiella pneumoniae in said sample.
 6. The method of claim 5,wherein amplification step comprises a method selected from the groupconsisting of: (a) polymerase chain reaction (PCR), (b) ligase chainreaction, (c) nucleic acid sequence-based amplification, (d)self-sustained sequence replication, (e) strand displacementamplification, (f) branched DNA signal amplification, (g) nested PCR,and (h) multiplex PCR.
 7. The method of claim 6, wherein saidamplification step comprises PCR.
 8. The method of claim 5, furthercomprising contacting the sample with a probe that hybridizes to aportion of the atpD gene amplified by said amplification primer pair. 9.The method of claim 8, wherein said probe comprises a fluorescentmoiety.